Tuyet Minh

Th nghiem phan lap va nuoi cay te bao ung th co t cung
at van e
S song qua phc tap khong cho phep ch qua mot vai nghien cu ma
hieu c toan bo c the con ngi Linus Pauling. Hien nay tren the gii cac
ng dung ky thuat cua hang loat cong trnh nghien cu nuoi cay cac te bao
ung th a va ang giup cac nha khoa hoc tng bc giai ap nhng van e c
ban trong sinh hoc phan t va ca nhng ieu b an trong c the con ngi. Cac
nha khoa hoc van ang tiep tuc tm kiem va kham pha ra nhng dong te bao
ong nhat ve mat di truyen. Cac te bao c chon loc nay la muc tieu cho viec
tien hanh cac lieu phap sinh hoc nham gop phan ngan chan can benh hiem
ngheo.
Khoa hoc ve nuoi cay te bao ung th co pham vi ng dung rat rong va
phong phu ca trong nghien cu khoa hoc va trong thng mai. Dong te bao ung
th c s dung nh mot vat lieu e san xuat ra cac c chat. Cac protein ngi
co the c phan lap trc tiep t viec nuoi cay te bao. Cac dong te bao ong
vai tro quan trong trong cac ky thuat tai to hp DNA trong sinh hoc phan t.
Ngi ta con s dung dong te bao e th nghiem cac loai thuoc co oc tnh e
ieu tr cac benh di truyen, ung th. Viec nuoi cay te bao con gop phan m ra
ky thuat dung hp te bao. Ky thuat nay giup chung ta hieu biet nhng ieu b
an xay ra ben trong c the con ngi nh cac cau tao va chc nang cua gen
ngi, cac bieu hien va cac c che di truyen, cac phan ng sinh hoc bnh
thng va qua trnh phat trien cua nhng gen benh.
Muc tieu chnh cua luan van nay la gii thieu nen tang c ban va
phng phap luan can thiet e tien hanh nuoi cay te bao ung th noi chung va
ac biet la th nghiem phan lap va nuoi cay s cap te bao ung th co t cung
t cac mau mo sinh thiet cua cac benh nhan c chan oan b ton thng
loan san giai oan IB, IIA va IIB.
Viet Nam viec nuoi cay cac te bao con het sc mi me. Da tren cac
tai lieu co c vi s hng dan cua cac Thay Co, cac anh ch va s ho tr cua
benh vien Ung bu thanh pho Ho Ch Minh em a co gang tien hanh cong
viec nuoi cay dong te bao ung th co t cung trong pham vi kha nang, thi gian
va cac ieu kien ky thuat hien co. Ket qua cung mi ch dng mc o xac
nh te bao ung th co t cung co the nuoi cay trong mot moi trng nhat nh
vi cac thao tac chuan, kem theo ieu kien moi trng va cac dung cu th
nghiem can phai c vo trung tuyet oi. Mac du ty le thanh cong lan th
nghiem phan lap va nuoi cay au tien cua em la 1/20 mau sinh thiet nhng
ay la tien e e em co the tiep tuc theo uoi e tai nay trong tng lai.
Tp Ho Ch Minh, thang 7 2004
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1 Ngoc Tuyet Minh
1.1. MOT SO MOC LCH S NGHIEN CU VE UNG TH

Theo cac tai lieu c ghi chep t thi co ai, cac nha khoa hoc a tm thay dau
vet cua mot khoi u ac tnh mau xng hoa thach cua xac p Ai Cap (cach ay
5.000 nam) va mot ban thao c viet tren giay co lau mo ta 8 trng hp b khoi u
hay vet loet phan ngc. Trong ban mo ta ngi viet con ghi them khong co cach
ieu tr. Luc bay gi thuat ng cancer cha c s dung.
Hyppocrate, ngi thay thuoc Hy Lap cung xac nhan ung th co the lam chet
ngi. Vao thi nay a s dung thuat ng carcinoma e mo ta hai loai ung th: loai
sui ra ngoai nhieu chan nh loai cua (carcinoma) va loai phat trien sau trong tht
(sarcom).
Tuy nhien mai en the ky 15, Giovanni Morgagni mi la ngi au tien thc
hien giai phau t thi mot benh nhan u xng vi muc ch tm hieu ban chat va
nguyen nhan benh. ay la nen tang e phat trien khoa hoc ve u bu (oncology).
Nam 1846, nha nghien cu benh hoc ngi c Rudolph Virchow mo ta te bao ung
th mau va a ra gia thuyet ve nguon goc ung th te bao. Ong cho rang tat ca cac
te bao, bao gom ca te bao ung th eu co nguon goc t mot te bao khac. Gia thuyet
cua ong vao thi o a rat tien bo va vai thap ky sau gia thuyet nay c chng
minh la ung.
au the ky 19, cac nha khoa hoc mi he m phan nao s b an cua ung th.
Nam 1911, Peyton Rous a kham pha c mot trong nhng virut gay ung th au
tien at ten la RSV. o la mot retrovirus (virut phien ma ngc) ma von di truyen
c cau tao bang RNA. Thi iem nay mot van e at ra cho cac nha khoa hoc la
lam the nao RSV co the nhap vao von di truyen cua te bao cau tao bang DNA. Nam
1960, Temin ngh ra rang RNA co the tao ra DNA nh mot chat men. Mi nam
sau, Temin va Baltimore a chng minh c s hien hu cua enzym nay, o la
enzym sao chep ngc va cac virut gay ung th loai RNA c goi la virut phien
ma ngc. Kham pha nay nhan c giai thng Nobel Sinh ly va Y hoc nam 1975.
Nam 1976, nha nghien cu ngi Phap Dominique Stehelin cung vi cac ong
nghiep ngi My Michel Bishop va Harold Varmus kham pha mot ieu gay sng sot
la nhiem sac the cua nhieu ong vat, ke ca ngi cha cac gen co the chuyen oi
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thanh cac gen gay u, nh vay xac nh tren 60 loai gen cua te bao co the chuyen
thanh gen gay ung th.
Kien thc cua nhan loai ve di truyen hoc, ve c che sinh hoc phan t a tien
trien nhanh trong nhng nam gan ay.Vao gia the ky 20 cac nha khoa hoc mi bat
au giai quyet nhng van e phc tap cua di truyen hoc. James Watson va Franci
Crick vi hai trang thong bao ngan gon ve mo hnh phan t cua DNA tren tap ch
Nature a lam ao lon nhan thc cua gii khoa hoc ng thi. Trong lch s sinh
hoc t co moc nghien cu nao co tac ong rong rai nh mach xoan kep DNA cua ho.
Mo hnh cua Watson va Crick em lai y ngha mi me cho viec nghien cu chu e
di truyen hoc va ung th.
Cung vao thi iem nay thuat ng khang the n dong xuat hien. Ky nang tao
cac khang the n dong a cho ra i mot cong nghe mi ap dung pho bien trong
viec chan oan sm va ieu tr mot so benh ung th. Nha bac hoc tm ra cac khang
the n dong c trao giai thng Nobel vao nam 1981 la Cesar Milstein.
Chung ta phai tha nhan rang en cuoi the ky 20, nhan loai a co nhng bc
tien vt bac ve viec nghien cu ung th tren c s di truyen hoc, ong thi tm ra
nhng lieu phap ieu tr mi ma muc tieu nham vao viec tieu diet cac te bao ung
th chuyen hoa. Tuy nhien, nhng ieu nay en nay van cha lam thay oi s that
ve mot can benh nan y. Nhng du sao chung ta cung co quyen hy vong en nam
2010 hay lau hn na ban phan loai ve gen con ngi se c hoan tat, o se la nen
tang e cac nha khoa hoc tm ra cac tn hieu ieu khien qua trnh tai tao khoi u va
tm ra cac gen bao ve c the chong lai can benh nay.
1.2. NHNG AC TNH CUA UNG TH NGI

1.2.1. Ban chat cua benh ung th va s phan biet gia u lanh vi u ac tnh
Ung th la mot benh ly ac tnh. V sao ung th lai lam cho ngi ta that s s hai
khi mac phai? Ta co the hnh dung te bao ung th nh mot sinh vat phat trien ngoai
vong kiem soat. Ma cai g hoac ieu g nam ngoai vong kiem soat eu ang lo ngai.
Trong phan hoa te bao bnh thng, cac c che kiem soat mc te bao thng
han che sinh trng va phan bao nhng mc xac nh. Nhng oi khi mot te bao
thoat khoi mc kiem soat nay va nhan ban len mot cach thai qua. Sinh trng thai
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3 Ngoc Tuyet Minh
qua co the dan en hau qua tao ra khoi u, tc la mot khoi te bao bat thng so vi
nhng mo chuan mc bnh thng.
Co phai tat ca khoi u eu la ung th khong? cau tra li la khong. Co hai loai u. U
lanh tnh thng goi la u lanh. o la mot khoi te bao bat thng van gi lai v tr
goc ni chung a phat sinh trong c the. Cac u lanh cung co the gay s co neu
chung sinh trng ln chen ep mot so c quan b khung xng boc kn nh nao hay
phoi. Nhng thong thng cac v tr khong chen ep cac mo khac ngi ta co the
dung phau thuat tai cho cat bo hoan toan khoi u ra khoi c the.
Trai han vi u lanh la cac u ac tnh la mot khoi mo bat thng co the phat trien
lan vao trong cac mo lan can va thng di can vao nhng bo phan xa khac cua c
the. Mot u ac phat sinh t mot te bao ung th duy nhat va di cho ra khoi mo bnh
thng no a phat sinh. Neu khong tieu diet hay cat bo, mot vai te bao ung th o
se xam nhiem vao trong cac mo bao quanh va khoi u goc se banh trng. Cac te
bao cung co the tach khoi khoi u, xam nhap vao he tuan hoan (vao cac mach bach
huyet va mach mau) va co the lan en cac v tr mi, tao ra nhng khoi u mi tai
nhng cho o. S di cho cua cac te bao ung th ra khoi v tr nguyen thuy cua
chung c goi la s di can (malignant progression).
Hnh 1: S di can
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Noi chung, e phan biet gia u lanh tnh va u ac tnh co the da tren cac ac
iem biet hoa va mat biet hoa, ty le phat trien, tnh chat xam nhap tai cho va s di
can.
Thuat ng biet hoa va mat biet hoa ap dung cho cac te bao bieu mo cua khoi u.
S biet hoa e ch mc o phat trien cua cac te bao bieu mo giong cac te bao bnh
thng ca ve hnh thc va chc nang. U biet hoa cao la u gom nhng te bao giong
cac te bao cua mo sinh ra u. Noi chung cac u lanh tnh eu biet hoa cao.
Cac u ac tnh xep t biet hoa cao en khong biet hoa. Cac u ac tnh gom nhng
te bao khong biet hoa c goi la mat biet hoa (anaplasia). S mat biet hoa c
coi la dau an cua chuyen dang ac tnh. S mat biet hoa c anh dau bi mot so
thay oi ve hnh thai va chc nang nh ca te bao va nhan te bao eu bieu hien a
hnh thai thay oi ve kch thc va hnh dang. Cac te bao co the ln hn nhieu lan
nhng te bao ke can cung loai trong khi cac te bao khac co the cc ky nho, dang
nguyen thuy.
Mot hnh anh khac cua s mat biet hoa la s tao thanh nhng te bao khong lo,
mot so te bao co mot nhan a dang khong lo duy nhat va mot so khac co hai hay
nhieu nhan. Trong te bao khong lo ung th, nhan te bao tang sac va ln so vi te
bao. Ngoai cac bat thng ve te bao hoc mo ta tren, s phan cc cua cac te bao mat
biet hoa b roi loan ro ret. Cac dai hoac cac khoi ln te bao u phat trien vo to chc.
Mac du cac te bao ang phat trien nay oi hoi c cung cap mau, nhng do mo
em huyet quan ngheo nan nen vung trung tam u thng b hoai t v thieu mau.
1.2.2. Sinh san cua te bao ung th

1.2.2.1. S bat thung cua nhan va cac nhiem sac the
Trong cac te bao ung th co the bieu hien cac dang bat thng cua phan chia
nh khong co thoi vo sac co the dan en chet te bao hoac nhieu nhan chia nho.
Nhan chia vi nhiem sac the cc co ac iem la con ton tai trong gian ky, mot vai
nhiem sac the khong c huy ong en gan mot hoac hai trung the co the dan en
chet te bao hoac tao thanh hai nhan co so nhiem sac the khong eu. Tuy nhien
nhng dau an ac hieu cua te bao ung th thng bieu hien s bat thng nhiem
sac the.
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5 Ngoc Tuyet Minh
Cac phng phap xet nghiem gan ay con cho phep nhan biet nhng bat thng
nhiem sac the cua cac dang u khac nhau. V du nh nhiem sac the Ph1 quan sat thay
trong benh bach cau loai tuy man tnh. Cac u nguyen bao vong mac la do s thieu
hut mot phan nhiem sac the 13. Phan ln cac trng hp u mang nao thieu hoan
toan hay mot phan nhiem sac the 22.
1.2.2.2. Quan niem n dong

Ngay nay ngi ta cho rang mot ung th phat trien t mot te bao chuyen dang.
Thc ra ngi ta khong the xac nh mot ung th tren vi the trc khi no a tao
thanh nhieu te bao, nhng co nhieu bang chng chng minh rang ung th la mot
quan the te bao ong nhat, mot dong te bao. V du nh s che tiet globulin mien
dch n dong trong cac u tng bao va trong cac u lympho te bao B, tat ca cac te
bao u ch che tiet mot dang duy nhat cua globulin mien dch.
Ngi ta cung thay rang hien tng ghep mot te bao duy nhat co the co hieu qua
trong mot so ung th thc nghiem (benh bach cau chuot). Trong qua trnh tien trien
cua u, quan the te bao ung th chu nhng ot bien mi. S tien trien dong nay bieu
hien bi s xuat hien nhng chung bat thng hn, co tnh chat xam lan hn va e
khang hn vi cac bien phap ieu tr.
1.2.2.3. Thi gian nhan oi u

Thi gian nhan oi u la thi gian trung bnh can thiet e tang gap oi so lng te
bao ung th. Ngi ta co the o tren nuoi cay hoac tnh tren vivo t khoi lng u.
Thi gian nhan oi la mot thang cho nhng u phoi, hai en ba thang cho nhng ung
th bieu mo dang bieu b, bon en sau thang cho nhng ung th bieu mo dang
tuyen, bon en mi hai thang cho nhng u tuy.
Co moi lien he gia thi gian nhan oi va s phat trien cua u. Thi gian nhan
oi ngan u phat trien nhanh, thi gian nhan oi dai u phat trien cham, khong co thi
gian nhan oi u ngng phat trien. Trong phan ln thi gian phat trien khoi u, thi
gian nhan oi luon hang nh va se cang dai hn khi u a phat trien ln.
Thi gian nhan oi trc het phu thuoc vao he so tang sinh, he so mat te bao.
He so tang sinh bieu lo ty le cac te bao trong chu ky. He so mat te bao bieu lo ty le
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6 Ngoc Tuyet Minh
phan tram cac te bao b mat i do hoai t hoac do bong. Cung can phai nhan manh
rang cac mo bnh thng s mat te bao la 100%, so te bao sinh ra khong nhieu
hn te bao mat i o la s can bang noi mo cua mo. Trong ung th he so tang sinh
te bao thng tang cao nhat trong khi s mat te bao cung tram trong nhat. Trong cac
dang ung th, cac u mat biet hoa nhat cung la cac u co toc o phat trien va he so
tang sinh cao nhat.
Trong cac te bao ung th thi gian trung bnh cua mot chu ky te bao la mot en
ba ngay, pha S co thi gian kha hang nh (trung bnh 16 gi) nhng pha G1 hay
thay oi nhieu hn. Chu ky cua te bao ung th khong ngan hn chu ky cua cac te
bao bnh thng, tham ch cham hn do s keo dai cua phan bao nguyen nhiem. V
du chu ky cua cac nguyen bach cau cua benh bach cau cap la 50-60 gi, trong khi
chu ky cua cac nguyen bao tuy bnh thng la 24 gi.
1.2.3. Nguyen nhan gay ra ung th

Ngay nay cuoc chien chong ung th tren the gii c tien hanh toan dien tren
nhieu mat. Nhng nghien cu thc nghiem va dch te a ong gop ln lao trong viec
xac nh mot so nguyen nhan chu yeu cua ung th ngi. Cac nha khoa hoc a
chng minh ro rang rang s phat trien ung th thng la ket qua t s tiep can vi
nhieu yeu to rui ro trong moi trng song.
Hien nay, theo anh gia cua IARC (International Agency for Resarch on Cancer1998) co 75 tac nhan c ket luan gay ung th cho con ngi. Nhom tac nhan gay
h hai ve di truyen va gay chuyen dang u cua te bao thuoc cac loai sau ay: (1) chat
gay ung th hoa hoc, (2) nang lng tia, (3) vi khuan sinh ung th, chu yeu la cac
virut. Moi nhom tac nhan co ac iem rieng, nhng nhieu tac nhan co the cung tac
ong va lam tang hieu qua cac tac nhan khac.
Cac tac nhan gay ung th lam the nao e gay c ung th. Nh chung ta a biet
hau het ung th la ket qua t nhieu dang thay oi di truyen. Cac thay oi nay la ket
qua t hang chuc nam tiep xuc vi cac tac nhan ung th co anh hng gay ot bien.
Noi chung khi ty le phan bao cang cao th xac suat sinh ot bien cang cao do he qua
t de sinh nham lan trong t nhan oi va to hp lai DNA. Mot vai tac nhan gay ung
th dng nh co ca hai tac dung o. Chang han cac hocmon gay ra ung th vu va
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7 Ngoc Tuyet Minh
ung th t cung va gay ra tang phan bao va co the gay ra cac thay oi di truyen se
dan en ung th.
Nhng nam gan ay cac nha khoa hoc a nghien cu va biet c nhieu tap tnh
hoat ong gay benh cua cac te bao ung th. Nh viec nhiem virut viem gan B keo
dai co the sinh ung th gan. Nhiem virut u nhu (papilloma) gay ra u nhu bo phan
sinh duc co the dan en ung th co t cung. Virut gay ung th tr thanh mot dang
thng tru trong cac te bao vat chu, thng do chen nhan acid nucleic vao trong
DNA cua cac the nhiem sac vat chu.
Theo Zibbert, virut sau khi xam nhap te bao a lam thay oi ac tnh di truyen
cua te bao gay roi loan phan bao. Do o te bao tang san hon loan va mang nhng
ac tnh di truyen mi. ben trong te bao, virut a mat het hnh thai bnh thng
nen khong c phat hien na. Chat mau cua acid nucleic cua virut a hoa nhap ket
hp vi chat mau cua te bao, mot DNA c hnh thanh mang tnh chat cua te bao
ung th. Te bao benh o tiep tuc nhan len ma khong can s hien dien cua virut na.
Thong ke cac ca ung th lien quan en virut chiem khoang 15% cac dang ung th
ngi tren toan the gii.
Tuy nhien con rat lau chung ta mi biet het c cac nhan to a gop phan gay
ung th. Cac nha khoa hoc ang tiep tuc tm kiem nhng tac nhan b an nay bang
phng phap dch te hoc.
1.3. C S PHAN T CUA UNG TH

Tat ca sinh vat song eu c cau tao t te bao. Mot so loai sinh vat, v du nh
vi khuan, co the ton tai nh sinh vat n bao. Nhng sinh vat khac, bao gom ca ong
vat hu nhu c cau tao t vo so te bao, tat ca te bao ket hp vi nhau e tao nen
mot sinh vat song. Nhieu te bao cau tao thanh mo nh mo lien ket, mo c, nhieu mo
cau tao thanh c quan nh gan, phoi. Ngoai ra c the ngi con phu thuoc vao
nhng te bao nho chuyen biet, chung se cau tao nen nhng c quan chuyen biet nh
tim hay phoi. Nh vay, s hoat ong cua te bao giup duy tr s song. Tuy nhien
trong trng hp ung th, s thay oi chc nang va cau truc cua te bao la nguyen
nhan chnh.
1.3.1. Cac gen gay ung th (oncogen)

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Theo quan iem hien nay ung th c xem la benh cua gen bi v ngi ta phat
hien ra hang loat cac gen gay ung th (oncogen) va gen c che ung th
(antioncogen) co trong te bao. Khi cac gen nay b bien d th de dan en ung th.
Gan 50% cac ung th a c nghien cu, chan oan bang s bien d cua mot gen la
gen p.53. P.53 la mot antioncogen hay con goi la gen c che ung th.
Cac nha khoa hoc a chng minh rang trong c the lanh manh cua con ngi co
cha tien gen kch thch s phat trien (growth promoting protoocogen) hay con goi
la tien gen gay u. o la gen can thiet cho viec hai hoa s song.
Vai tro chuan mc cua cac tien gen gay u trong te bao la g? Nhieu tien gen gay
u ma hoa cho cac nhan to loan sinh trng cac protein kch thch phan bao hoac
ma hoa cho cac protein khac anh hng en tong hp hay chc nang cua cac nhan
to sinh trng. Khi chung the hien chc nang bnh thng, ung lng phu hp, vao
ung luc can thiet th cac protein nay kiem soat phan bao va kiem soat s phan hoa
ung chuan mc.
e mot tien gen gay u chuyen thanh gen gay u phai co mot thay oi hoac mot
ot bien xay ra trong DNA cua te bao. Co the la do mot nhiem sac the b chuyen
oi v tr. S chuyen v xay ra khi cac nhiem sac the trao oi lan nhau cac mang nho
cua chung. Th du s bien hnh ac tnh trong benh bach cau tuy th lien quan en s
chuyen v tr protooncogen t v tr bnh thng the nhiem sac 9 len the nhiem sac
22. o thc ra la mot nhiem sac the phoi hp gom than la nhiem sac the so 9, tren
o mot manh cuoi cua nhiem sac the 22 gan vao. Protooncogen myc trong ung th
lymphoma Burkitt la do chuyen v tr the nhiem sac 8 len 14.
mot trng hp khac la s ot bien tren DNA, mot nham lan trong t nhan oi
DNA hoac trong tai to hp phat sinh khi nhan ban cac gen a c sao ma hay dch
ma toan bo, hau qua la qua tha protein kch thch phan bao so vi mc chuan.
Trong cac trng hp nay mc bieu hien gen chuan b thay oi va te bao b kch
thch sinh soi nay n mot cach tuy tien.
Hien nay cac oncogen tao ra cac protein tham gia kiem tra s ln len cua te bao
co the c phan loai theo chc nang nh sau:
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Nhom th nhat la gen ma hoa cho cac yeu to tang trng nh sis, int-2. Nhng
oncogen yeu to tang trng nay co the kch hoat s nhan len cua te bao nhng
khong u kha nang gay nen thay oi ve kieu hnh..
Nhom th hai cua oncogen ma hoa cho cac thu the cua yeu to tang trng nhieu
thu the trong so o gan vi hoat tnh tyrosine kinase.
Nhom th ba la cac thu the khong co hoat tnh tyrosine kinase o la san pham
gen mas.
Nhom th t la cac ho protein gan tren mang nh ras.
Nhom th nam la nhng oncoprotein trong te bao chat vi hoat tnh
serine/threonine kinase gom co san pham raf, pim-1, mos va cot.
Nhom th sau gom cac oncogen ma hoa cho cac yeu to ieu hoa trong te bao
chat.
Nhom th bay, mot nhom oncogen ln ma hoa cho nhng yeu to phien ma
nhan nh myc, myb, fos, jun, rel.
1.3.2. Cac gen c che ung th (antioncogen)

Trong khi cac tien gen u ma hoa cac protein khi ong s phat trien te bao, th
cac san pham cua cac gen c che khoi u co chc nang la ieu hoa s phat trien te
bao. S ot bien hay mat gen c che khoi u co the gay ra ung th. Trong benh bu
nguyen bao vong mac, mot loai ung th cua tre em mang tnh di truyen, gan ay cac
nha khoa hoc a kham pha ra c nguyen nhan la do mat mot oan cua nhiem sac
the 13. oan b mat o mang cac gen kiem che khong cho cac oncogen cua ung th
nay phat trien.
Them nhieu bang chng xac nhan rang bat ky ot bien nao ngan chan gen triet
khoi u eu gop phan gay loan phat phan bao, tc khong kiem soat noi mc phan
bao chuan mc va co the sinh ra mot te bao ung th.
ung th ai trang nh nhieu dang ung th khac ngi, ngi ta nhan thay
qua trnh di can cua mot ung th ai trang phat trien t t qua ba chang. Dau hieu
au tien la trong cac te bao bnh thng cua bieu mo lot ai trang xuat hien tan so
phan bao bat thng. Sau o xuat hien mot u lanh (choi polip) trong vach ai trang.
Va cuoi cung xuat hien mot khoi u ac tnh. Nhng thay oi mc te bao nay dien ra
Ta
10 Ngoc Tuyet Minh
song song vi ba thay oi mc o DNA bao gom s hoat hoa cua mot gen gay u
trong te bao va s vo hieu hoa cua hai gen c che khoi u.
1.3.2.1. Gen Rb
Gen lam phat trien bu nguyen bao vong mac (retinoblastoma) ngi c
goi ten la gen Rb (retinoblastoma supectability gene). ot bien gen Rb lam phat
trien dang ung th khac ngi nh sacom xng (osteosarrcom). Khac vi benh
retioblastoma do di truyen, nhng ot bien cua Rb thung xuat hien te bao sinh
dng hn la do di truyen t bo me. Retinoblastoma tao ra do ot bien hai lan
pham vi gen Rb cua the nhiem sac 13.
Thong qua cac nghien cu ngi ta biet san pham protein cua gen Rb tham gia
trong ieu hoa tang trng te bao.
1.3.2.2. Gen p53

Gen nay nam tren nhanh ngan cua the nhiem sac 17. Protein p53 co vai tro ngan
can s lan tran cua cac te bao b h hai ve di truyen. Protein p53 khu tru trong
nhan te bao va khi tac ong, no co chc nang au tien la kiem soat s phien ma ca
cac gen khac. S bien oi p53 xay ra gan mot na so ung th ngi. P53 c
huy ong e lam ngng s h hai DNA va ho tr cho viec sa cha DNA bang cach
gay dng G1 va kch thch cac gen DNA c sa cha. Mot te bao co DNA b h
hai neu khong c sa cha se c p53 ieu khien i vao chet te bao theo chng
trnh.
Do cac hoat ong nay p53 ang c goi la ngi canh gac bo gen. Neu mat
ong hp t cua gen p53, ton hai p53 khong c sa cha, ot bien tr thanh co
nh trong cac te bao ang phan chia va te bao se chuyen dang sang ac tnh, ac
biet la mo lien ket.
Gen p53 a c phat hien gan 20 nam nay va c xem la gen duy nhat gi vai
tro trung tam trong c che gay benh ung th.
1.3.2.3. Gen p73
Ta
11 Ngoc Tuyet Minh
en cuoi nam 1977, cac nha nghien cu phat hien ra gen p73, gen nay ma hoa
mot protein mang nhieu ac iem tng t nh gen p53. Gen p73 co mot vung gan
DNA giong mot vung tng ng cua p53, no co the gay dng chu ky te bao va gay
chet te bao theo chng trnh trong nhng ieu kien thch hp.
Nhieu nghien cu hien nay ang tap trung vao gen ho hang cua p53 nay. Gen
p73 pho bien trong nhieu loai u nh u nguyen bao than kinh, ung th ai trang.
1.3.3. Gen ieu hoa chet te bao theo chng trnh
Chieu xa
Bax
Chemos
p53
Apoptosis
Bcl-2
Oncogens
p21
E1A, tTAg,
Gadd45
Chu trnh te bao
c-myc
S o 1: Hien tng chng trnh hoa s chet te bao
Apoptosis la hien tng chng trnh hoa s chet cua te bao (programmed
cell death PCD) hien tng nay tr nen can thiet trong giai oan phat trien nao
o cua te bao. Ngi ta cung quan sat thay hien tng nay trong ung th nh giet te
bao ung th bang te bao NK (natural killers) hay bang te bao oc cung nh te bao
lymphocyt oc phu khang the. Nhng te bao ung th de i vao apoptosis la khi gay
ra DNA h hai c quyet nh s co mat trong chuyen dang bien d cua gen p53
hoac s dung cac thuoc pha G1 hoac cac tia pha G2.
C che ieu hoa vong te bao pha p53 cha c biet chnh xac nhng co le no la
chat ieu hoa sao ma cua cac gen tham gia photphoryl hoa nhieu cho qua nhieu
enzym kinaza, v du kinaza cdk1 (cell division kinase) lam photphoryl hoa protein
p53, p73 v tr Ser-315. Khi c photphoryl hoa, p53 cung co anh hng en cac
protein bien dang cua cac virut gay ung th loai DNA. Chnh cho nay p53 co the
Ta
12 Ngoc Tuyet Minh
ong vai tro chu yeu trong ieu hoa ln len, biet hoa va s chet te bao theo chng
trnh nh san.
S bieu hien gen cua protein p53 c ieu hoa qua nhieu khang nguyen, trong
o khang nguyen PCNA (proliferation cells nuclear antigen). Khang nguyen nay la
cofactor cua enzym polymeraza sao chep S. Bi vay khi giam hoat tnh p53 th
phong be s i vao pha S cua chu ky phan chia te bao.
Cuoi cung, ngi ta phat hien protein km ham hoat tnh cac kinaza cdk khac
nhau c goi la CKI (cyclin dependent kinases inhibitors) va quan trong nhat trong
chung la p21 san pham cua gen nh 1 (p53 mediated inhibitor of cdk).
Mot chat c che kinaza cdk khac nhau la protein p16 INK 1 (Inhinitor of cdkS),
gen ma hoa no khu tru trong nhieu dong te bao ung th. Gen nay km ham nhieu
ung th mang ten MTS 1 (multiple tumor suppressor 1) hay CDK 4 I (CDK 4
inhibitor).
Kien thc co lien quan ti c che apoptosis te bao ung th rat can trong nghien
cu ieu tr ung th. Cung can nh rang gen p53 co the hp tac vi gen Rb trong
cam ng apoptosis va bien hnh ung th.
Hieu biet ve gen p53 va gen Rb bnh thng, ngi ta co the tach dong (cloning)
chung e dung trong ieu tr ung th, o la mot trong nhng van e hap dan cua
sinh hoc phan t.
Protein p.53
Kch thch tong hp p.21

(c che cdk)
Phong be
Sao chep AND
Kch thch gen c che
Km ham GTP
Km ham gen ieu hoa
tang trng dng tnh
tang trng te bao

Lam ngng sinh san te bao giai oan G
S o 2: Vai tro cua Protein p.53
Ta
13 Ngoc Tuyet Minh
Ngay nay a xac nh c rang cac gen e phong hay gay chet te bao theo
chng trnh cung tham gia tao nhng bien oi quan trong trong s can bang ung
th. Mot nhom ln cac gen ieu hoa chet te bao theo chng trnh a c xac nh,
cac gen nay c ghi nh bang cac nhom ba ch bat au bang ch b.
Gen chong chet te bao theo chng trnh lan au tien a xac nh, bcl-2 la mot
thanh phan cua mot nhom ln protein homodime va heterodime hoa, mot so protein
nay c che te bao chet theo chng trnh (nh chnh bcl-2 va bcl-xL) trong khi cac
gen khac (nh bax, bad, va bcl-xS) tao ieu kien cho te bao chet theo chng trnh.
Viec phat hien ra gen bcl-2, mot gen prototyp trong loai nay bat au vi nhan
xet la khoang 85% cac u lympho te bao B cua loai nang co mang mot chuyen oan
ien hnh t(14:18)(q22:q21). Can nhac lai rang 14q32 la v tr cac gen chuoi nang
cua globulin mien dch cung lien quan en u lympho Burkitt. Viec xep canh v tr
hoat ong phien ma vi bcl-2 (khu tru 18q21) gay boc lo qua mc bcl-2.
Theo nhng c che cha biet ro, viec boc lo qua mc bcl-2 bao ve cac lympho
bao khoi b chet theo chng trnh va cho phep chung song lau dai, v vay gay nen
s tch luy qua mc cua lympho bao B, gay benh hach va xam nhap tuy xng. V
cac u lympho boc lo qua mc bcl-2 phan ln phat sinh la do giam chet te bao theo
chng trnh hn la do bung no tang sinh te bao, no co xu hng lanh tnh (phat
trien cham) so vi phan ln cac u lympho khac.
Chng minh cho vai tro cua bcl-2 trong tao u lympho la nhan xet rang chuot
chuyen gen vi bcl-2 phat sinh u lympho te bao B. Khong ch chc nang cua bcl-2 la
bat thng ma s khu tru cua no cung khac vi hau het cac gen ung th khac. No
nam la ngoai cua mang ty the, li noi nguyen sinh va mang nhan. S nh v ty
the cua gen bcl-2 va cac thanh vien khac cua ho bcl-2 cung bieu hien chc nang cua
cac gen nay.
C s sinh hoa cua hoat ong bcl-2 cha c hoan toan hieu biet ro. Chet te
bao theo chng trnh la iem cuoi cung cua chuoi cac s kien phan t gay nen bi
nhieu kch thch va ve sau dan en s hoat hoa cac men thuy phan protein gay chet
te bao (men caspase). Cac thanh vien cua ho bcl-2 anh hng mot cach chnh xac
ti s hoat hoa cua cac caspase nh the nao ang c khao sat tng tan.
Trong nhieu mo hnh chet te bao theo chng trnh, viec giai phong cytochrom C
khoi ty the la bc quan trong trong chuoi cac s kien dan en chet te bao theo
Ta
14 Ngoc Tuyet Minh
chng trnh. Mot chc nang cua cytochrom C uc giai phong hnh nh la ho tr
cho s hoat hoa men caspase 9 phan huy protein. Khu tru mot cach chien lc
mang ngoai ty the bcl-2 va yeu to tng ong cua no c cho la co vai tro ieu hoa
s i ra cua cytochrom khoi nhiem sac the vao trong bao tng te bao.
S i ra cua cytochrom C nay c ieu hoa mot cach chnh xac nh the nao th
cha c biet ro, nhng co mot so bang chng la bax, mot thanh vien trc chet te
bao theo chng trnh cua ho bcl-2 tao thanh mot kenh trong mang nhiem sac the
cho phep s i ra cua cytochrom C (va v vay dan en chet te bao theo chng
trnh), trong khi bcl-2 phong toa hoat ong tao kenh cua bax.
Cac thanh phan trc chet te bao theo chng trnh va chong chet te bao theo
chng trnh cua ho bcl-2 tac ong nh mot cai bien tr trong ieu hoa chet te bao
theo chng trnh. Ty le cua cac chat chong chet (bcl-2, bclXL) vi chat gay chet
(bax, bcl-xS, bad, bid) se quyet nh mot te bao ap ng vi mot kch thch chet te
bao theo chng trnh nh the nao. Cai bien tr nay se hoat ong t nhat mot phan
do s dime hoa canh tranh gia cac thanh phan khac nhau cua cac nhom chat. V
vay trong khi cac homodime cua bcl-2 tao ieu kien cho s song sot cua te bao (co
le do s thay the bax tao kenh t mang ty the), cac homodime bax tao thuan li cho
chet te bao theo chng trnh.
Cuoi cung mac du ho bcl-2 cua cac gen bcl-2 ong vai tro quan trong trong ieu
hoa chet te bao theo chng trnh, t nhat co hai gen ket hp vi ung th khac cung
lien quan en chet te bao theo chng trnh la gen p53 va protooncogen c-myc. C
che phan t cua chet te bao theo chng trnh gay nen do hai bc cat ngang cua
ng bcl-2. Gen c-myc gay chet te bao theo chng trnh khi cac te bao b ieu
khien bi hoat hoa c-myc, nhng s phat trien c gii han bi kha nang gii han
cua cac yeu to moi trng.
Khi oi mat vi cac tn hieu nh vay, te bao c lap chng trnh chet bi ieu
hoa tang p53 va cac tn hieu cha c xac nh khac. Bieu lo qua mc cua bcl-2 co
the giai thoat cho te bao khong b chet theo chng trnh gay nen do c-myc. V vay
hnh nh myc va bcl-2 co the hp tac trong tao u - c-myc kch thch sinh san va bcl-2
ngan can chet te bao ngay ca khi cac yeu to phat trien tr nen b han che. ay la
mot trong nhieu v du trong o hai hay nhieu gen ung th hp tac e gay phat sinh
ung th.
Ta
15 Ngoc Tuyet Minh
1.4. MOT SO PHNG PHAP TACH VA NUOI CAY TE BAO UNG

TH
1.4.1. Lch s phat trien nuoi cay cua te bao ung th
Khoa hoc ve nuoi cay mo va te bao a phat trien mot cach vng chac trong suot
the ky 20. T nhng nam 1885, Wilhelm Roux ghi nhan rang phoi ga co the phat
trien dung dch muoi sinh ly trong vai ngay. Nhieu th nghiem trc o cung
chng minh rang mo cua cac loai ong vat lng c co the tai sinh trong qua trnh
nuoi cay. Nam 1887 Arnold cho rang cac lympho bao cua ech cung co the song
trong dung dch muoi sinh ly. Ngay sau o vao 1998 Ljunggren chng minh te bao
da cua ngi co the phat trien neu c nuoi cay trong dung dch asetic.
Mac du a co nhng nghien cu trc nhng ngi c xem la cha e cua
phng phap nuoi cay mo la Ross Harrison. Harrison lay mo t phoi ech a vao
hach bach huyet th cac manh mo khong ch co kha nang song sot ma con phat trien
thanh cac mang li. Cac th nghiem tren bc au a tao nen lan song chu y ve
nuoi cay mo in vitro, ong thi s thuc ay cua y hoc buoc ngi ta quan tam en
cac loai vat on nhiet va co qua trnh phat trien bnh thng va benh ly gan giong
ngi.
Nam 1943, Wilton Earle - ngi au tien tien hanh th nghiem ve dong te bao
lien tuc loai gam nham t mot te bao tach ri. Nam 1951, Geogre Gey thiet lap
dong te bao ung th t co t cung ngi (Hela). Nam 1961, Hayflick va Moorhead
a thc hien nhng nghien cu ve te bao bnh thng co i song xac nh.
1.4.2. Mot so phng phap tach te bao ung th t khoi u

1.4.2.1. Phng phap tach te bao ung th t khoi u cng hay khoi u di can
Hien nay e co the thu nguon mau ung th, ac biet la t cac khoi u cng hay
khoi u di can t c the benh nhan b ung th eu phai c tien hanh phau thuat tai
cac benh vien. Nguon mau sau khi thu se c gi trong bnh hoac lo cha san moi
trng nuoi cay thch hp (nh RPMI 1640, DMEM ), ong thi phai c gi
Ta
16 Ngoc Tuyet Minh
trong ieu kien lanh nh p a, hoac tot nhat la c gi trong nit long v ieu
nay se giup duy tr c ac tnh te bao.
Qua trnh thu mau va x ly mau phai c tien hanh nhanh chong. Mau sau khi
thu se c tien hanh x ly tai phong th nghiem. Qua trnh x ly mau phai c
thao tac trong ieu kien vo trung. Sau khi la chon phan mo con song va loai bo
phan mo h hoac chet, phan mo song con lai se c ra qua nhieu lan bang dung
dch muoi sinh ly co cha khang sinh hoac dung dch HBSS Hanks balanced salt
solution e loai bo bt mau. Sau o cat khoi mo ra thanh tng manh nho (1-2 mm3),
thng c tien hanh tren a petri. Sau khi them vao a petri mot t dung dch
nuoi cay (1 2ml), dung pipet hoac pastette e chuyen cac manh mo t a petri
vao bnh nuoi cay, e trong tu nuoi khoang t 2 3 gi, ieu nay se giup cho s
bam cua te bao vao bnh nuoi cay tot hn. Them vao 5 10ml, e yen bnh nuoi
cay cho en t 72 96 gi. Sau nhieu ngay nuoi cay, te bao phat trien bam dnh vao
thanh bnh va khi en mat o can thiet se c tien hanh cay chuyen.
ay la mot trong nhng phng phap t lam ton thng en te bao nhat, thi
gian thao tac ngan, ong thi de dang thao tac. Nhng ieu can lu y la thao tac
phai chuan, nhng mau mo phai c cat nho, qua trnh th nghiem phai c tien
hanh trong ieu kien vo trung tuyet oi e tranh s nhiem.
1.4.2.2. Phng phap tach te bao ung th t mau dch cua c the
Phng phap nay thng c s dung e tach te bao t nhng mau dch cua c
the nh: dch mang bung, dch mang phoi
Cac mau dch c thu tai benh vien va em ve x ly tai phong th nghiem vo
trung, tuy theo mau dch lay vi the tch khac nhau. em mau dch i ly tam,
khoang 3000g/ 20 phut. Loai bo dung dch noi, huyen phu phan can vi dung dch
muoi sinh ly PBS. Neu mau cha so lng te bao mau nhieu ta co the loai bo bang
phng phap ly tam vi dung dch Ficoll. Khoang 10ml dung dch te bao huyen phu
vi PBS hoac HBSS cho vao dung dch Ficoll, ly tam khoang 1000g/ 20 30 phut.
Cac te bao hien dien trong phan dung dch em, em ly tam khoang 600g/ 5 phut.
Ta
17 Ngoc Tuyet Minh
Qua trnh nay lap lai cho en khi thu c het phan te bao con lai. Toan bo phan te
bao thu c se c huyen phu vi moi trng nuoi cay va chuyen vao a nuoi.
1.4.2.3. Phng phap tach te bao bang enzym

S dung enzym e tach te bao t nhng mo song la mot trong nhng phng
phap pho bien nhat. Hien nay mot so enzym c s dung trypsin, collagenase,
hyaluronidase, elastase, dipase, va papain tuy thuoc vao mau mo e chon enzym
va nong o enzym thch hp nham co the tach c te bao va ong thi khong anh
hng en s phat trien cua chung.
Chang han trypsin la mot enzym c s dung pho bien trong viec tac te bao
ong vat vi hai quy trnh:
(1) Quy trnh trypsin am
-
Cat nho khoi mo thanh nhng manh nho khoang 2 3mm3.
Them dung dch trypsin vao (1g khoi mo/ 10ml trypsin), nong o trypsin thng
c s dung la 0,25% (hoac co the la 0,05%, 0,015%), hoac co the them vao
chat nen EDTA (0,01% -0,02%),.
Khuay t nhiet o 37oC. e at c mat o te bao can thiet th thi gian

khuay co the t 15 45 phut.
em i ly tam, 600g/ 5 phut, loai bo phan noi, them moi trng vao chuyen te
bao vao bnh nuoi cay.
Qua trnh nay lap lai cho en khi thu c het phan te bao con lai.
(2) Quy trnh trypsin lanh
Co the u cac manh mo nho trong dung dch trypsin nong o thch hp, tai nhiet
o khoang 4oC, ieu nay se giup cho viec tach te bao de dang va t anh hng en
te bao. Sau 6 8 gi, giai ong khoang 30 phut nhiet 37oC. Sau khi ly tam loai bo
phan noi, them vao moi trng nuoi cay.
1.4.3. Nhng ky thuat can thiet cho viec nuoi cay te bao
Ngay nay viec nuoi cay te bao a c thc hien rong khap tren the gii. Hn
50 nam ke t lan au tien cong bo s xuat hien dong te bao ung th co t cung
ngi (Hela), en nay cac nha nghien cu a thiet lap khoang 3.000 dong te bao
Ta
18 Ngoc Tuyet Minh
ung th va co 1000 dong c s dung thng xuyen, phan ln co nguon goc t ung
th ngi.
ay la nguon mau can thiet cho cac phan tch sinh hoa cua te bao ung th. ieu
nay m ra c hoi cho cac nha khoa hoc co ieu kien nghien cu, tm hieu sau hn
ve cac ac tnh sinh hoc ong thi xac nh va mo ta ac iem cua te bao ung th.
Vi muc ch s dung cac dong te bao nay e nghien cu hoat tnh cua cac hoat
chat khang ung th trong ieu kien in vitro, san xuat vaccin ieu tr.
Mac du nhng d lieu co c t cac phong th nghiem van can phai c xem
xet mot cach than trong, nhng vai tro thc tien cua viec nuoi cay cua cac dong te
bao ung th trong nghien cu va ieu tr benh ung th a c tha nhan.
1.4.3.1. Moi trng nuoi cay

Hien nay ngi ta s dung moi trng nuoi cay tong hp a c phat trien t
nhng nam 1950. Cac chat c ban co trong moi trng nuoi cay bao gom amino
acid, carbohydrat, Vitamin, va muoi.
Huyet thanh va cac thanh phan khac nh yeu to tang trng hocmon c them
vao moi trng c ban e cung cap nhng chat can thiet cho te bao phat trien. Moi
trng nuoi cay au tien la moi trng BME (Eagles basal medium), ay la moi
trng do Harry Eagle thiet lap co the s dung cho ca te bao bnh thng va te bao
khoi u. Moi trng phat trien tiep theo sau c goi la moi trng toi thieu MEM
(Eagles minium essential medium) cung do Harry Eagle thanh lap, MEM nay c
tang cng them ham lng amino acid. Trong khi o th moi trng DMEM
(Dulbeccos modified Eagles medium) do Dulbecco cai tien, trong o cac thanh
phan cua amino acid va Vitamin tang gap 4 lan, glucose tang 4,5 lan.
Moi trng IMDM (Iscoves modified Dulbecco medium) do Iscove thiet lap
tren c s cai tien moi trng DMEM. Moi trng nay cha selenium, amino acid,
vitamin, sodium pyruvate, dung dch em HEPES. Moi trng GMEM (Glasgow
minium essential medium) la s bien oi khac cua moi trng Eagle dung e nghien
cu cac yeu to gay anh hng en te bao. Moi trng nay co cha amino acid va
vitamin cao gap hai lan so vi moi trng Eagle.
Ta
19 Ngoc Tuyet Minh
Nam 1950, Morgan va ong s a cho biet moi trng co the ho tr cho cac mo
phat trien, o la moi trng 199, cha nhieu thanh phan co the tm thay moi
trng Eagle va co the s dung cho nhieu loai te bao khac nhau.
Mot moi trng khac t phc tap hn la CMRL 1066 c thiet lap tai Connaught
Medical Research Institue, ay la moi trng au tien khong s dung huyet thanh
nhng co bo sung them mot vai chat ho tr gan giong huyet thanh. Mot moi trng
khac khong huyet thanh c goi la moi trng Waymouth dung e nuoi cay te bao
L929 chuot, nhng moi trng nay cung c chng minh la co the s dung trong
nuoi cay cac dong te bao khac.
Nam 1959, McCoy mo ta cong thc c ban e tao ra moi trng ho tr cho muc
tieu nuoi cay s cap. Moi trng RPMI 1640 do Moore va ong s thiet lap tai Park
Memorial Institue, c s dung pho bien cho nhieu dong te bao khac nhau va cung
c cac phong th nghiem chon la e nuoi cay te bao va mo bach huyet.
Moi trng co the c cung cap t cac nguon thng mai vi cac cong thc
khac nhau, nh moi trng co nong o bnh thng (co the s dung t 9 12
thang), hoac moi trng co nong o 10X (co the s dung trong vong 12 24 thang),
hoac moi trng co nong o am ac (co the s dung t 2 3 nam).
1.4.3.2. Huyet thanh

Mac du moi trng co cha nhieu chat bo dng can thiet cho s phat trien,
nhng huyet thanh la yeu to ho tr cho kha nang song va phat trien cua te bao trong
nuoi cay. Huyet thanh kch thch s phuc hoi cac ton thng cua te bao khi cay
chuyen va cac protein trong huyet thanh lam bat hoat trypsin tranh cac enzym gay
ton thng te bao. Huyet thanh co tnh khang oxy hoa manh, ong thi chung con co
tac dung cai thien tnh dnh cua te bao len be mat bnh nuoi.
Huyet thanh bao gom hocmon, cac nhan to tang trng, lipid, cac protein van
chuyen va cac chat ket dnh. Trong cac nghien cu nuoi cay te bao ung th, huyet
thanh be va huyet thanh thai bo c s dung rong rai, ngoai ra cung the s dung
huyet thanh ngi va nga.
Hien nay, tr nhng dong te bao c thuan hoa vi moi trng tong hp hoan
toan, a so c nuoi cay trong moi trng co bo sung t 5 20% huyet thanh la tot
Ta
20 Ngoc Tuyet Minh
nhat va ty le nay tuy thuoc vao loai te bao nuoi cay. Neu s dung ty le huyet thanh
cao se lam tang gia thanh san pham.
1.4.3.3. Moi trng khong co huyet thanh

S dung huyet thanh cho phep cac te bao phat trien khi nuoi cay nhng cung co
nhng ieu bat li. au tien, mac du thanh phan cua moi trng a c nh ro
nhng lng huyet thanh co the thay oi tuy thuoc vao moi t nuoi cay. S thay
oi ve so lng huyet thanh se dan en s thay oi ve cac thong so bao gom ty le
song sot cua te bao, tien trnh phat trien. Mot vai thanh phan trong huyet thanh co
nhng yeu to c che s phan bao cua mot so te bao. Ben canh o mac du huyet
thanh c thng xuyen kiem tra nhng van co kha nang xuat hien virut.
V nhng ly do tren cung vi s can nhac ve gia thanh, nen a xuat hien cac
khuynh hng chuyen qua s dung moi trng khac. Vao nhng nam 1960 1970,
ngi ta a tm ra moi trng khong can s dung huyet thanh hay dung vi lng
thap. Sato va ong s la nhng ngi tien phong tm ra nhng chat ac biet hien
dien trong moi trng c ban e thay the huyet thanh. Trong khi o Ham va ong s
tang cng nong o trong thanh phan cua moi trng c ban bang cach bo sung
hocmon, cac protein van chuyen, lipid va cac nhan to ket gan.
1.4.3.4. Moi trng vat chat

Thong thng cac te bao ong vat phat trien tot nhat nhiet o 36,5oC + 1oC,
tuy the nhiet o thap hn te bao van phat trien, nhng chung se chet mot cach
nhanh chong khi nhiet o khoang 40oC. ac biet khi nuoi cay te bao ong vat
ieu kien ve o am va nong o CO2 trong khong kh rat quan trong. Nong o CO2
thng la 5% thch hp nhat.
Phan ln cac te bao ung th can thiet phai on nh o pH t 7,2 7,4 va moi
trng phai c thay oi thng xuyen. V khi te bao phat trien se tao nen s ho
hap gay ra acid hoa moi trng. Co the kiem tra o pH khi them phenol o vao moi
trng. Neu moi trng mau vang pH 6,5; mau cam pH 7; mau o pH 7,4; mau o
ta pH 7,8. o am khi nuoi cay cung rat quan trong, s boc hi t moi trng co
muoi se lam cho te bao b phan giai gay ton hai en mang te bao.
Ta
21 Ngoc Tuyet Minh
Hau het cac te bao ung th phat trien can thiet phai co gia va hau nh cac te
bao ung th phat trien t lp n tren a thuy tinh hay a nha. Mot so trng hp
ngoai le la dong te bao tao mau va te bao ung th phoi nho thch hp phan chia
the huyen phu. Te bao bam dnh co the tang so lng ln bang cach bam vao cac
gia nh vao cac thanh phan chat nen (ECM Extracellular matrix). Hien nay cac
protein ECM c s dung pho bien bao gom collagen, fibronectin, laminin.
1.4.4. Nuoi cay s cap

Nuoi cay s cap la lan nuoi cay au tien cac mo c lay trc tiep t c the.
Nuoi cay te bao ung th co the lay cac mo t mot khoi u ran c the (t mo goc
hoac di can) hay la te bao the huyen phu, v du nh la hut dch te bao mang
bung hay mang phoi. ac biet cac dang te bao the huyen phu nay thuan li cho
viec phat trien dong te bao bi v trong dch a co san te bao n oc va cac cum te
bao. ieu nay tranh c s tach te bao bang phng phap c hoc hay s pha huy
cua enzym.
Cac thanh phan cau tao te bao lan nuoi cay s cap gom nhieu mau mo thng
khong ong nhat, co nhieu dang nh mo tao mau va cac dang te bao stroma, nguyen
bao si. Tuy nhien sau mot hay hai the he cac dong te bao nuoi cay co tnh chat
ong nhat hay t nhat cung dang. Cac dang te bao ban au b chuyen dang di ap
lc chon loc cua ieu kien nuoi cay e tao nen s ong nhat cua dang te bao manh
nhat. Do o moi lan cay chuyen mau se giong nhau va ac tnh dong te bao duy
tr qua nhieu the he.
Te bao c nuoi cay phai c kiem tra thng xuyen (tot nhat la moi ngay) ke
ca quan sat bang ai the va vi the. Hnh thai va mat o te bao c xem xet qua
knh hien vi va phai anh gia c s lay nhiem cua vi khuan va cac vi sinh vat
khac. Kiem tra bang cam quan ve mau sac, tnh chat moi trng (uc, day, hoac
m). Moi trng phai c thay oi hang ngay va khong cho phep e can moi
trng hay thieu cac chat dinh dng hoac b acid hoa. o pH cua moi trng
thng c kiem soat de dang bang phenol o.
1.4.5. Cay chuyen
Ta
22 Ngoc Tuyet Minh
Khi cac te bao c nuoi cay phat trien tren toan bo be mat cua bnh nuoi cay
(oi vi lp n) hoac la oi vi nuoi cay te bao the huyen phu thi iem ma
moi trng a suy yeu chat dinh dng luc o oi hoi te bao phai c cay chuyen
e nham duy tr kha nang phat trien cua te bao. Tuy nhien khi thc hien cay chuyen
th mat o te bao se giam xuong nhng mc o te bao co the phat trien tr lai mot
cach toi u nhat va am bao lng moi trng ay u cho te bao phat trien tiep tuc.
Ngi ta s dung enzym nh trypsin e tach te bao va phng phap tach nay
cung tac ong en ket qua thu hoach te bao trong nhng lan nuoi cay. Sau khi nuoi
cay s cap, te bao qua mot vai lan cay chuyen se c tien hanh nuoi cay th cap
e tao ra dong te bao.
Cay chuyen c thc hien tot nhat khi te bao phat trien log phase, luc o te
bao c xem la khoe manh nhat. Sau o dong te bao can phai c xac nh va
nhan dang nguon goc.
1.4.6. Tao dong

Dong la mot quan the te bao co nguon goc t mot te bao rieng biet va tao dong
la qua trnh tach te bao rieng biet va phat trien cac te bao the he sau. V vay tao
dong co kha nang duy tr phan bao trong mot thi gian dai ma khong thay oi ac
tnh ban au cua mo goc.
Hieu qua tao dong c anh gia tren tieu chuan nh kha nang nuoi cay, cac
dong c san xuat, so lng te bao c nuoi cay. Thng hieu qua tao dong trong
nuoi cay s cap rat thap khoang di 1%, trong khi o van co nhng dong te bao co
hieu qua tao dong cao hn trong nuoi cay s cap, tuy thuoc vao tng dong te bao ma
hieu qua nuoi cay se thay oi t 10 100%. Ngi ta co the tao dong te bao tren
a hoac trong agar.
1.4.7. Ky thuat ong lanh te bao

Bao quan nguon te bao di nhiet o 130oC cho phep d tr te bao trong mot
khoang thi gian dai t 20 en 30 nam. Thong thng ngi ta s dung chat xuc tac
ong lanh la DMSO (dimethylsulfoxide) hoac Glycerol.
Ta
23 Ngoc Tuyet Minh
Thng thi gian ong lanh hay ra ong cung anh hng en kha nang song cua
te bao va ong lanh 1oC/phut c xem la ap ng tot nhat. Ngc lai quy trnh ra
ong phai c tien hanh nhanh. ieu nay de dang c thc hien trong nc am
nhiet o 37oC. Thng te bao c tr trong nit long nhiet o -196oC, nhng co
the duy tr s song trong thi gian ngan - 80oC.
1.5. NHAN DANG DONG TE BAO UNG TH

1.5.1. Gii thieu
Theo cac tai lieu nghien cu hien nay co khoang 3.000 dong te bao ung th c
thiet lap va co 1.000 dong c s dung thng xuyen. V vay can thiet phai nhan
dang va tm hieu ac iem cua cac dong te bao ung th. Trong qua trnh nuoi cay
hnh thc bieu hien cua te bao co the thay oi va cac net ac trng nh tnh chat
biet hoa co the mat i, nhng ac tnh di truyen van duy tr qua nhieu the he.
Co nhieu ly do quan trong e can thiet xac nh ac iem cua cac dong te bao
ung th. ieu trc tien o la moi lien quan gia dong te bao ung th va mo goc,
can thiet phai xac nhan rang dong te bao ung th nay co nguon goc t mo goc, ieu
nay co gia tr ac biet trong viec xac nh cac lieu phap tr benh. ieu quan trong
th hai la nham kiem soat tnh ong nhat va kha nang lay nhiem gia cac dong te
bao vi nhau.
Lch s cua qua trnh nuoi cay te bao cho thay rang s lay nhiem gia cac dong
te bao van thng xay ra. Trong suot nhng nam 70-80 nhieu cong trnh nghien cu
cua Stanley chng minh rang s lay nhiem a xay ra dong te bao Hela. He qua la
dan en s lay nhiem lan tran cac phong th nghiem va s pha tron gia cac dong
te bao a xuat hien.
Van e lay nhiem hien nay van con la mot van e nan giai. Cac nha nghien cu
khong ch quan tam en s lay nhiem gia cac dong te bao cung loai ma con la s
lay nhiem gia cac dong te bao khac loai. V vay can thiet phai co phng phap
khong ch xac nh ro nguon goc duy nhat cua dong te bao ma con phai xac nhan
nguon goc cua loai.
Ta
24 Ngoc Tuyet Minh
Ngi ta a dung cac phng phap nh cac marker DNA, phan tch cytogenic,
PCR e xac nhan nguon goc cua dong te bao va kiem tra s lay nhiem vi cac
dong te bao khac. Mat khac s xac nhan ac iem con bao gom s kiem tra, theo
doi cac iem ac trng cua dong te bao, s quan sat hnh thai, ty le phat trien, hieu
qua tao dong, cac bieu hien khang nguyen, ac iem chu trnh te bao.
1.5.2. ac iem phat trien cua dong te bao ung th

ieu quan trong cua dong te bao ung th la kha nang cung cap nguon te bao mi
e tien hanh cac nghien cu ve benh ung th. Dong te bao phai phan anh tnh chat
cua mo goc.
Hien nay, gan nh tat ca cac te bao ung th co the phat trien trong nuoi cay. Hau
het dong te bao ung th co kha nang phan chia khong gii han, oi vi cac dong te
bao lien tuc th kha nang phan chia len en ca ngan lan. Mot dong te bao co the tr
thanh dong te bao lien tuc (thay v gii han) mac du te bao hien dien mc o thap
trong lan nuoi cay au tien nhng nhng te bao nay phai co kha nang phan chia
khong gii han, hoac la chung phai trai qua qua trnh bien nap. S bien nap tao nen
bi mot virut cam ng ung th hay bang hoa chat.
Khi cay chuyen, cac dong te bao ung th se trai qua nhieu giai oan phat trien
nhat nh. Lp te bao n giai oan cay au tien se bam vao gia va khi c
tach ri bi enzym (nh trypsin) th can phai co thi gian e te bao hoi phuc lai
nhng h hai do s pha huy cua enzym. thi iem nay te bao phat trien tng oi
cham, c goi la lag phase pha tiem ot bien.
Khi viec nuoi cay tiep tuc cong vi s thay oi cac yeu to tang trng trong moi
trng th se lam tang kha nang phan chia te bao va lam tang so lng te bao. ay
la giai oan phat trien nhanh, c goi la log phase, thng te bao phat trien theo
luy tha.
Cuoi cung khi te bao khong con tiep xuc vi chat nen hoac lien quan en viec s
dung lng huyet thanh hay cac thanh phan khac trong moi trng luc o mat o te
bao se giam xuong, ngi ta goi o la s bao hoa mat o hoac goi la plateau
phase, ay la mot trong nhng tnh chat cua dong te bao trong ieu kien nuoi cay
ac biet.
Ta
25 Ngoc Tuyet Minh
Thng ngi ta ghi nhan rang trc khi cay chuyen plateau phase dai hn,
con sau khi cay chuyen th lag phase dai hn.
1.5.3. Xac nhan ac iem dong te bao ung th

Khi mot dong te bao mi c thiet lap moi quan tam hang au la phai xac nh
ro nguon goc cua dong te bao. Thong thng ngi ta nuoi cay dong te bao t khoi
u nguyen phat, t dch c the hoac t khoi u di can Khi xac nh nguon goc te bao
neu ch xet tren kha canh nay se khong co gia tr mot cach hoan toan. Gan ay,
ien hnh la s ch nh lai dong te bao SW626, theo cac nha khoa hoc rat co the la
dong te bao nay co nguon goc t ung th ket trang hn la t ung th buong trng (t
nhng nam 1974 ngi ta xac nh SW626 la dong te bao co nguon goc t ung th
buong trng).
Hien nay cac nha khoa hoc cho rang cac d lieu bao gom c s mo hoc, giai
oan va mc o biet hoa cua khoi u, bien phap ieu tr (xa tr, hoa tr) can thiet phai
c xem xet khi xac nh nguon goc dong te bao. Cac d lieu nay khong ch ch li
cho viec chan oan va ieu tr benh ong thi con cung cap vat lieu cho cac marker
DNA, hay ac iem cua te bao nham so sanh vi dong te bao mi phat trien.
Trong qua trnh nuoi cay phai ghi chep ay u cac mc o phat trien cua te bao,
ac biet la cac ch so ve dong te bao.Thng xac nh ac iem cua dong giai
oan sm th tot hn. Khi nuoi cay te bao giai oan sm (nuoi cay s cap) thng
co cha nhieu loai te bao hon hp. V du khi nuoi cay te bao ung th bieu b co the
cha cac te bao bieu mo va cac loai te bao khac nh nguyen bao si, ai thc bao.
V vay khi nuoi cay cac te bao nay se di ong va ngi ta co the de dang co c
quan the te bao ong nhat.
Them mot ieu can lu y na la dong te bao mi thiet lap phai c at ten (e
tranh s nham lan vi cac dong te bao khac va yeu cau phai gi kn b mat ve ten
ngi cho). Hien nay cac nha nghien cu khong s dung ten benh nhan e at ten
cho dong te bao v rat de xay ra s nham lan. Thng ngi ta at ten cho cac dong
te bao theo cach xac nh ni san xuat va nguon goc mo.V du nh dong te bao ung
th buong trng co ten goi la SKOV3 (Sloan Kettering Ovarian Cancer). ieu nay
Ta
26 Ngoc Tuyet Minh
rat co li v ten goi cua mot dong te bao mi xuat hien tren th trng se cung cap
mot lng thong tin ban au kha ro va chnh xac cho cac benh vien, thay thuoc.
1.5.4. Tap hp cac dong te bao ung th

Hien nay tren the gii a thanh lap To chc Hiep hoi The gii ve nuoi cay te
bao trong o co tren 62 quoc gia la thanh vien cua To chc nay, tap hp khoang 469
nhom. Rieng dong te bao ung th co nhieu nhat ATCC (American Type Culture
Collection). ATCC co khoang 950 dong te bao ung th trong o co 700 dong te bao
co nguon goc t ngi.
Hien nay chnh phu cua cac nc nay ang co ke hoach ho tr vo ieu kien cac
hoat ong tren. Cac ngan hang te bao cac to chc tren co kha nang cung cap
nhng dong te bao tot nhat cho cac hoat ong nghien cu cac phong th nghiem,
cac trng ai hoc, benh vien. Cac ngan hang bao am nguon te bao khong b
nhiem (ac biet la nhiem Mycoplasma). ay c xem la yeu to hang au e anh
gia mc o uy tn va danh tieng cua cac ngan hang cung cap dong te bao.
Hien nay co mot van e c at ra cho cac ngan hang te bao la phat trien them
cac dong te bao mi hay s dung cac dong te bao hien co. Tren thc te cac nha
khoa hoc nhan nh rang khong co thi gian e phat trien them nhng dong mi.
Chung ta ch co the co quy thi gian e s dung nhng dong te bao a co mot cach
co hieu qua nhat. Cac nha khoa hoc cho rang can phai co nhieu no lc va danh
nhieu thi gian nhat tap trung vao viec tm ra nhng ac iem cua cac dong te bao
ung th e co phng phap tr benh ung th mot cach co hieu qua nhat.
1.6. Cac ng dung cua ky thuat nuoi cay te bao ung th

1.6.1. Khang the n dong
Thuat ng n dong co ngha la moi te bao san xuat ra cac khang the eu la con
chau cua mot te bao duy nhat, do o chung san xuat mot loai phan t khang the
ong nhat.
Cac khang the n dong c thu t nhng te bao nuoi cay ch khong phai thu
t cac ong vat thc nghiem. o la san pham cua cac te bao lai tao gia mot te bao
Ta
27 Ngoc Tuyet Minh
ung th ac tnh von co the nhan ban vo tan trong moi trng nuoi cay vi mot te bao
B san xuat khang the chuan. Te bao lai mang tnh bat t cua te bao ung th va mang
kha nang san xuat ra cac khang the cua te bao B. Ngi ta co the tach rieng tng te
bao lai va nuoi rieng re. Moi te bao se nhan len thanh mot dong te bao thuan khiet
ve moi mat ke ca tnh ac hieu vi mot khang nguyen.
Cac khang the n dong co kha nang tieu diet cac te bao ung th tren c the
ngi va tren chuot mang ung th thc nghiem. Chang han mot khang the n dong
chuyen hoa cho mot khang nguyen tren te bao ung th co the to hp vi mot chat
thuoc diet cac te bao. Khi tiem chung vao mot benh nhan ung th, chuyen tnh cua
khang the se nham muc tieu la cac te bao ung th va phan thuoc cua to hp se diet
cac te bao ung th o. Phng phap tiep can e ieu tr ung th nay ang c th
nghiem nhieu va co ket qua ang khch le trong cac ca ung th tuy xng.
1.6.2. Cac Interferon (INF)

Interferon la protein c cac te bao nhiem vi rut san xuat ra e giup cac te bao
khac khang virut. C che hoat ong cua interferon nh sau:
(1) cac virut nhiem vao cac te bao
(2) bat cac gen interferon trong nhan cac te bao e sao mARN thong tin
(3) thuc ay te bao che tao cac interferon
(4) te bao nhiem virut se chet nhng cac phan t interferon cua no co the
khuyech tan sang nhng te bao khoe manh lan can kch thch chung sinh san ra cac
protein khac e khang virut va khang khoi u.
Co ba loai interferon chu yeu la INF, va , trong o INF c s dung rong
rai nhat va co hoat tnh ro ret trong benh bach cau te bao toc, bach cau man tnh the
tuy, benh a u tuy va mot so u lympho ac tnh khong Hodgkin. Ngoai ra cung co tac
dung gii han trong mot so ung th than, ung th hac to, sarcom Kaposi va mot so
ung th bieu mo khac.
1.6.3. Cac hoat chat khang ung th

Ky thuat nuoi cay mo con la oi tng trong cac nghien cu ve sinh hoc te bao,
nh s dung e nghien cu cac hoat chat khang ung th trong ieu kien in vitro.
Ta
28 Ngoc Tuyet Minh
Hien nay ngi ta s dung mot so thuoc ngan chan cac te bao ung th phan chia
c goi la thuoc khang nguyen phan, ngan nga phan bao bang can thiep ti thoi
phan bao.
Mot th thuoc loai nay co ten la vinblastin, phan huy cac vi ong, do o ngan
chan hnh thanh thoi t. Ngay nay vinblastin c nhieu cong ty thuoc bao che t
loai cay da can, mot loai cay hoa mau hong ti, la co hoat chat vinoblastin, v
ang, tnh mat, c s dung di dang muoi sunfat ieu tr ung th.
Mot th thuoc khang nguyen phan khac la taxol, lam cac vi ong bat ong, do o
ngan thoi t tien hanh thay oi cac ong thai phan bao can thiet. Taxol co nhieu ha
hen hn v so vi nhieu loai thuoc chong ung th khac, no gay t phan ng phu va
dng nh co hieu lc ca vi dang ung th buong trng va ung th vu kho ieu tr.
Taxol chiet t vo cay thuy tung Thai Bnh Dng, mot loai cay moc nhieu
vung Tay Bac Hoa Ky. Cay thuy tung khi b eo vo se lam cay chet. Va c ieu tr
ung th cho mot benh nhan can phai huy diet t nhat ba cay o mot tram tuoi. Cuoi
cung taxol tr thanh mot chu e tranh cai cua cac nha khoa hoc bao ve thien nhien.
Gan ay cac nha nghien cu a ap dung cac tien bo ky thuat s dung nhieu loai
thuy tung khac nhau, tong hp nen taxol trong phong th nghiem va nuoi cay te bao
thuy tung thay v phai trong thuy tung trong moi trng t nhien. Va mi ay cac
nha khoa hoc cung a tm c mot loai nam moc tren thuy tung t chung cung co
the tao ra mot lng nho taxol. Ngi ta hy vong trong mot vai nam na nhng tien
bo ve cong nghe sinh hoc se giup con ngi khong can chat ha cay thuy tung Thai
Bnh Dng mi che tao c loai thuoc taxol nay.
1.7. NHNG VAN E LIEN QUAN EN E TAI THC HIEN

1.7.1. S lc ve ung th co t cung
Ung th co t cung (cervical carcinoma) la mot trong nhng benh ung th hang
au va la nguyen nhan th hai gay ra t vong cho phu n tren toan the gii. Hang
nam co 371.000 ca benh mi chiem ty le 10% trong tat ca cac benh ung th phu
n.
Ta
29 Ngoc Tuyet Minh
Cac vung co ty le cao nhat la Trung va Nam My, mien Nam va ong Chau Phi
va vung Caribe vi ty le mac phai t nhat la 30 ca mi tren 100.000 phu n moi
nam. ong Nam A chiem ty le trung bnh vi gan 20 ca mi tren 100.000 phu n
moi nam. Viet Nam, ung th co t cung cung la mot benh pho bien. Theo thong
ke ve ghi nhan ung th quan the thanh pho Ho Ch Minh (1998) a co them 28,6
ngi tren 100.00 phu n mac benh nay moi nam, mot ty le kha cao so vi khu vc.
S o 3 : Ty le mac phai va t vong cua benh ung th co t cung cac khu

vc do To chc y te The gii (World Health Organization WHO) kiem soat.
Ung th co t cung khi phat t lp bieu mo cua moi sau, lo trong hoac cua moi
trc co t cung. Giai oan au thng khong co hnh anh ac biet, khi phat trien co
ba hnh dang ai the khac nhau nh dang choi sui, dang loet, dang cng tham
nhiem. Dang vi the thng gap la ung th bieu mo te bao vay (squamous cells
carcinoma) chiem 85%. So con lai la ung th bieu mo tuyen.
Ta
30 Ngoc Tuyet Minh
a
c
Hnh 2: Co t cung (a: co t cung; b: co t cung sach; c: co t cung b sui)
Hien nay cha co cach nao phong nga hu hieu loai ung th nay ngoai viec
sang loc te bao hoc nh ky e phat hien va ieu tr kp thi cac ton thng loan san
hay tan san noi bieu mo co t cung (cervical intraepithelial neoplasia CIN). Tan
san noi bieu mo co t cung c xep t o I en III. CIN I tng ng vi loan san
nhe, CIN II bao gom loan san nang va ung th bieu mo tai cho. CIN III c xem la
ton thng tien ung th vi nguy c cao tien trien thanh ung th bieu mo xam nhap.
Vien Ung th quoc gia My e ngh thuat ng chan oan mi gom co hai nhom
ton thng: nhng ton thng trc ay goi la CIN I c xep vao loai ton thng
noi bieu mo o thap (low- grade squamous intraepithelial lesion LSIL) va nhng
ton thng CIN II va III c xep vao loai ton thng noi bieu mo vay o cao (highgrade squamous intraepithelil lesion HSIL).
Ta
31 Ngoc Tuyet Minh
TE BAO BIEU MO
UNG TH BIEU MO
(CARCINOMA)
MACH
BACH
HUYET
MO
LIEN KET
C TRN
MACH MAU
TE BAO DI CAN
SARCOMA
(BU THT)
Hnh 3: Hnh thai va s phat trien ung th bieu mo

1.7.2. Cac hnh thai benh ly co t cung
1.7.2.1. Ton thng viem
Co hai loai ton thng viem thng gap la viem do nam va viem do trichomonas
vaginalis. Quan sat tren nen phien o viem do nam thay co bach cau a nhan t
nhieu thoai hoa, co cac si nam candida albicans co hnh ot tre hay chia nhanh nh
cac canh cay hoac cac si nam leptothrix manh dai nh si toc. Viem do tricomonas
vaginalis th thay co rat nhieu sinh vat n bao dai 10-30 micromet, bao tng hnh
qua le, mat roi hay ch co mot phan roi, co nhan lech mot ben hay khong ro nhan
nhng de phat hien chung do mau xanh xam khac vi mau en cua nhan te bao
viem. oi khi ben canh cac ton thng do viem co the thay ton thng d san, loan
san i kem.
Ta
32 Ngoc Tuyet Minh
1.7.2.2. Ton thng d san

Ton thng d san la do s bien oi bieu mo tru co t cung thanh bieu mo dang
vay nhng khong co s thay oi ve cau truc va hnh thai te bao tren mo hoc. Nhng
te bao d san ien hnh co the ng rieng le hoac sap xep thanh nhom, am, kch
thc tng ng cac te bao can ay nhng phan ln hnh a dien, co moi lien ket
mat thiet, xep thanh nhieu tang nh te bao vay da, co the thay cac uoi bao tng
hay cau noi te bao nhng khong thay c chat sng.
1.7.2.3. Ton thng loan san

Ton thng loan san co s bat thng ve cau truc lan hnh thai te bao cua bieu
mo vay nhng mc o khac nhau. Trong a so trng hp loan san nhe khi quan
sat tren nen phien o thay cac te bao ay nho hn te bao trung gian hoac ngoai vi,
chung ng rieng le hoac xep thanh tng am nho, hoac nhom te bao tha tht vi
hnh thai te bao tron hay a dien, tnh a hnh nhe. Bao tng khong rong, co the
cha nhng hoc sang nho.
Trng hp loan san va, te bao loan san va nho hn te bao trung gian hay
ngoai vi nhng nhan khong eu, tang sac va thng ln hn trong loan san nhe. Van
co the thay hoc sang trong bao tng nhng mc o khac nhau va khong nhan
c hat nhan to, noi ro. Nhng te bao nay co the ng rieng le, xep thanh tng dai
kieu dong chay, noi uoi va khi thanh am th mc ao lon cau truc cung ro
hn loan san nhe bieu th bang cac te bao co lien ket long leo va hng lon xon.
Moi quan he gia virut sinh u nhu ngi (HPV) va condylom c anh gia
nh loan san nhe hoac ton thng bieu mo vay o thap. Cac condylom c ac
trng ve te bao hoc bang cac te bao bong (koilocyte). Nhng te bao nay to hn
nhng te bao d san, ng rieng le, xep thanh nhom hoac thanh am vi cac ac
iem la te bao khong eu hoac hai nhan, co nhng vung sang khong eu quan nhan
va mang bao tng va co ac va day len ro ret.
Trng hp loan san nang cac te bao co tnh chat khong ien hnh ro, trong o
loai can ay va trung gian sau chiem u the so vi cac te bao trung gian nong va te
bao ngoai vi cung co loan nhan. Thng nhan te bao sam mau, to nho khong eu,
li nhiem sac ac. Co the thay nhng nhan co mui, hnh nhan chia trong khi bao
Ta
33 Ngoc Tuyet Minh
tng hep, u baz va le ng nhien ty le nhan tren bao tng tang len. Trong
vung loan san nang co the thay cac nhan co mui, nhan quai va nhan chia.
1.7.2.4. Ung th tai cho

Y van ban rat nhieu ve loai ton thng nay nhng noi chung eu thong nhat kho
phan biet vi loan san nang ve te bao hoc. Nhn chung ac iem ac tnh bieu hien
ro hn nh te bao u ay, can ay co vien bao tng mong, u baz, nhan c ln,
khong eu nhau ro, tang sac, co the co thuy, mui, goc canh, ty le nhan va bao tng
b ao ngc, chat nhiem sac ket tu thanh cuc ln, khong eu co khi en nh mc.
Co the thay hat nhan ph ai.
1.7.2.5. Ung th bieu mo vay xam nhap

Ve mo hoc cung nh te bao hoc, ngi ta co the phan loai ba tp te bao ung th
co t cung: tp te bao to khong sng hoa, tp te bao to co sng hoa va tp te bao nho.
Quan sat te bao ung th co t cung trong nuoi cay hoac tren nen phien o thay co
nhieu te bao viem, manh vun te bao, hong cau thoai hoa va chat hoai t (nhat la
hoai t huyet) nam lan vi nhieu te bao ung th co khi day ac khap ni hoac co khi
ng rai rac thanh tng am, bo, to nho khong eu nhng kch thc te bao va nhan
thng khong ln lam.
Loai te bao ung th sng hoa nhan khong eu, tang sac ro, co the co nhan quai
va net ac trng la bao tng u acid v co chat sng. oi khi co kem rai rac te bao
hnh thoi vi nhan dai, sam, cung co the thay te bao u rat to.
Loai te bao ung th khong sng hoa co tnh a dang ro, nhan tron, hnh bau duc.
Te bao u ay thng nho hn loai sng hoa, ng rieng le hay tu tap nh hp
bao.
Loai te bao ung th nho co te bao gan giong nh te bao mam cua bieu mo vay,
chung nho nhng nhan to, tang sac, bao tng hep, co the thay hat nhan nhieu, to,
noi ro, chat nhiem sac bat thng.
1.7.3. HPV va moi lien he vi ung th co t cung
Ta
34 Ngoc Tuyet Minh
Moi lien he gia HPV xam nhiem ng sinh duc vi ung th co t cung a c
mot nha virus hoc ngi c, Harold zur Hausen, nhac en au tien vao nhng nam
1980. Ke t o, nhieu nghien cu quy mo ln ve cau truc phan t cua virut va c
che gay benh cung nh nhng van e ve dch te hoc a c thc hien nhieu
nc. Tam quan trong cua moi lien he nay tham ch con cao hn lien he gia hut
thuoc la va ung th phoi.
e cung co them bang chng ve viec HPV gay ung th, mot so nghien cu cho
thay khi nhiem HPV vao dong te bao nuoi cay, virut lam cho cac te bao phat trien
mot cach thieu kiem soat theo kieu ung th va cac te bao nay thc s gay ung th
khi tiem vao chuot.
Benh ung th co t cung xay ra phu n trong tuoi sinh san va nh cao la
o tuoi t 35 en 55 tuoi. Ngoai vai tro quan trong cua HPV, benh con chu tac ong
cua nhieu nhan to khac nh viec hut thuoc la, quan he sinh duc vi nhieu ngi, co
nhieu con, uong thuoc nga thai, he mien dch bat thng hay cac benh truyen
nhiem khac. ac biet la trang thai mien dch bat thng cung la mot nhan to quan
trong ay manh nguy c ung th co t cung khi co s hien dien cua mot hay vai loai
HPV.
S o 4: S o mo hnh nguyen nhan gay nhiem HPV va ung th co t cung

(mo ta vai tro cua nhng nhan to hanh vi anh hng en kha nang nhiem HPV
va lam phat trien ung th co t cung).
Ta
35 Ngoc Tuyet Minh
Human papilloma virus (HPV) thuoc ho Papovaviridae, la mot nhom virut c

biet en do gay ra mot so dang mun coc, u nhot (papilloma) hoac cac mun nhot bo
phan sinh duc. Trong nhieu nam lien, cac mun nhot c xem nh vo hai, ngoai tr
van e tham my. Tuy nhien trong thc te, co nhieu type HPV lien quan trc tiep en
nhieu benh khac nhau t mun coc thong thng cho en ung th.
Hien nay, ngi ta a phat hien hn 100 type (loai) HPV, trong o co hn 30
type chu yeu xam nhiem ng sinh duc, nhieu type a c giai trnh t. HPV
chu yeu xam nhiem cac te bao bieu mo, ac biet ng sinh duc. Chu trnh cua
HPV khac vi hau het cac virut khac cho s xam nhiem xay ra nhng te bao noi
b hoac bieu b c van con kha nang tang sinh.
Bang 1: phan loai HPV
Khoi u noi bieu mo co
Dang HPV
t cung
Khong xac nh
30, 34, 39, 40, 53, 57, 59, 61, 62, 64, 66, 67, 68,
69
Low risk
6, 11, 16, 18, 31, 33, 35, 42, 43, 44, 45, 51, 52,
74
High risk
16, 18, 6, 11, 31, 34, 33, 35, 39, 42, 44, 45, 51,
52, 56, 66
Ung th co t cung
16, 18, 31, 45, 33, 35, 39, 51, 52, 56, 58, 66, 68,
70
Gia nam 1974 va 1976, cac nha nghien cu bat au at van e va phan tch vai
tro cua cac type HPV trong ung th co t cung. Nam 1976, Meisels va Fortin cong
bo hai bao cao cho rang s hien dien cua koilocyte (te bao dang mat cu do s co rut
cua nhan va mot vang trong m xung quanh nhan gay ra bi s xam nhiem cua cac
thanh phan papillomavirus) trong mau quet te bao co t cung cho thay co s xam
nhiem bi HPV. Ho cung cho rang co the phan biet gia cac u nhot khong phat trien
thanh ung th co t cung va nhng khoi u tien ung th. Y tng nay c cung co
nh viec xac nh nhng hat t cua papillomavirus trong nhng khoi u bat thng
co t cung.
Ta
36 Ngoc Tuyet Minh
Cac mo ta ve tnh khong ong nhat cua ho papillomavirus va s co lap c mot

so kieu papillomavirus t mun nho vung sinh duc, cung nh viec ap dung lai phan
t mc t nghiem ngat e xac nh cac type HPV lien quan a cho mot cai nhn ro
rang ve s lien he gia nhiem HPV vi ung th ng sinh duc, ac biet la s co lap
va tao dong HPV 16 va HPV 18 t mau sinh thiet ung th co t cung.
Nhieu nghien cu sau o a cho nhieu hieu biet tot hn ve chc nang cua gen
virut oc va chi tiet qua trnh xam nhiem bi HPV. Nam 1996, Hoi Y te the gii
(World Health Association) cung vi To chc nghien cu ve xam nhiem va ung
bu ng sinh duc Chau Au (European Research Organization on Genital
Infection and Neoplasia) va Hoi ngh thong nhat cac Vien Sc khoe quoc gia ve ung
th co t cung a cong nhan HPV la nguyen nhan quan trong dan en ung th co t
cung. C quan quoc te ve nghien cu ung th cua To chc Y te the gii a xep loai
xam nhiem HPV la gay ung th oi vi ngi la HPV 16 va HPV 18, hau nh chac
chan gay ung th la HPV 31, HPV33 va co the gay ung th la cac type HPV khac
(tr HPV6 va HPV11). Do o, hien nay xam nhiem HPV c coi la nhan to trung
tam gay nen ung th co t cung.
1.7.4. Cau truc, thanh phan va hoat ong cua HPV
Hnh 4: Hnh anh au tien ve thanh phan cua cac virut qua knh hien vi ien
t c Strauss va ong s cong bo nam 1949.
HPV co ng knh khoang 55 nm cha DNA dang vong, mach oi, lien ket
vi protein giong histone. Vo capsid c tao thanh t 72 n v capsomere, moi
n v la mot pentamer cua protein cau truc L1. Ngoai ra con co mot loai protein
cau truc khac la L2 co thanh phan khang nguyen lien quan en cac phan ng vi
huyet thanh mien dch ac hieu 3,6.
Ta
37 Ngoc Tuyet Minh
1.7.4.1. C che gay benh

S phat trien ung th do Papillomavirus thng co s sat nhap bo gen virut vao
bo gen cua te bao chu, v tr sat nhap la bat ky. Thong thng bo gen virut se c
m vong gia khung oc m cua E2, lam bat hoat gen nay. Mot phan cua E2 va
cac gen khac gan ke vi E2 nh E4, E5 va mot phan cua L2 thng b mat sau khi
sat nhap. oan gen sat nhap luon gi vung ma hoa va ieu hoa cua gen virut E6,
E7.
Trong bo gen virut, gen E2 c biet la ma hoa cho nhan to ieu hoa theo c
che ieu hoa am promotor ieu khien s phien ma cua gen E6 va E7. Khi sat nhap
vao bo gen te bao chu, gen E2 khong bieu hien chc nang dan en s bieu hien
vt mc gen E6 va E7 va protein gay ung th c dch ma.
Nhieu bang chng c a ra cho thay vai tro quan trong cua gen E6, E7 cua
virut:
- Gen E6/E7 luon c bieu hien trong te bao ung th.
- S vang mat cua nhan to c che gen E6/E7 trong te bao ung th.
- Te bao ngi c bat t hoa khi co s hien dien cua cac gen nay.
- E6/E7 antisense RNA lam giam s phan chia te bao trong dong te bao lien tuc
va lam mat tnh trang ung th.
- Protein E6 va E7 c bieu hien vt mc sau khi sat nhap bo gen virut vao te
bao chu. Chung co kha nang gan ket va vo hieu hoa chc nang cua protein ieu hoa
tang trng te bao trong chu trnh te bao va c xem la nguyen nhan dan en tnh
trang ung th.
Ta
38 Ngoc Tuyet Minh
HPV
S NHIEM TIEM TANG
LP BIEU MO
TE BAO AC TNH
SAN SINH S NHIEM
GAN p53
va pRb
HPV
E6 / E7
S SAT NHAP HPV DNA
AP NG
MIEN DCH
Hnh 5 : C che gay benh
1.7.5. Protein virut va chc nang

1.7.5.1. Protein E1
E1 co mot so nhng ac tnh tng t nh khang nguyen T ln cua virut SV40,
no ma hoa cho mot RNA polycistron, mot protein co chc nang gan ac hieu vao
DNA, gan va thuy phan ATP, co hoat ong thao xoan khong phu thuoc ATP va rat
thiet yeu cho s sao chep cua papillomavirus. No cung co tng tac vi DNA
polymerase (alpha) te bao. Ben canh L1, khung oc m cua E1 la cau truc bao ton
nhat gia cac type virut khac nhau.
1.7.5.2. Protein E2
Khung oc m cua E2 ma hoa t 2-3 protein khac nhau, tat ca eu la nhan to
phien ma. Chung co nhng anh hng khac nhau en s bieu hien gen cua virut va
thc hien ieu hoa trong bo gen bang cach tao dimer nhng v tr gan thch hp.
Trong nhieu trng hp, protein E2 co kha nang c che s phien ma cua protein
virut E6 va E7. Protein E2 cua HPV 16 va HPV 18 co chc nang cua mot yeu to
hoat hoa phien ma te bao sng co t cung ngi.
Ta
39 Ngoc Tuyet Minh
S mat gen E2 thng thay trong sinh thiet ung th co t cung va trong dong te
bao cua loai ung th nay, cho thay s mat oan nay tao thuan li cho s chuyen
sang trang thai ac tnh. Khong ch ot bien trong khung oc m cua E2 ma ca nhng
ot bien trong vung DNA gan E2 trong LCR cung dan en tang cng hoat ong
gay ung th cua HPV 16. Trong qua trnh phat trien ung th, s t gay E2 la mot
hien tng xay ra muon v trong nhng ton thng tien ac tnh khong co hien tng
nay. Ngoai chc nang hoat hoa phien ma, E2 con tng tac vi E1 de dang gan vi
iem khi au sao chep.
1.7.5.3. Protein E4
San pham cua gen E4, protein E4, ong vai tro trong s trng thanh va phong
thch HPV. Qua trnh nay khong lam tan te bao.
1.7.5.4.
Protein E5
Protein E5 quan trong trong giai oan au cua s xam nhiem. No hoat hoa s
phat trien cua te bao bang cach tao ra cac phc hp vi receptor cua cac nhan to
tang trng va biet hoa. Ngoai ra, protein nay con co vai tro trong viec ngan chan
s chet theo chng trnh cua cac te bao co DNA h hong.
1.7.5.5. Protein E6
Protein E6 cua HPV16 cha 151 amino acid va cha 4 cau truc gan kem ieu
hoa Cys-X-X-Cys co the tao thanh hai cau truc ngon tay kem. Protein E6 cua HPV
nguy c cao co mot so tnh chat sinh hoc ang chu y nh lien ket vi protein E7
trong viec bat t hoa te bao ngi, tham ch protein nay co the bat t hoa mot so
loai te bao ong vat hu nhu ngay ca khi khong co s hien dien cua protein E7.
Protein E6 cua nhng virut nay lien ket vi gen ras trong qua trnh bat t hoa te bao
c ban cua chuot va kch thch s phat trien cua te bao
Tnh chat quan trong nhat cua protein E6 la no co kha nang gan vao protein p53
cua te bao. S gan ket nay lam tang s phan giai cua p53 bi he thong phan giai
Ta
40 Ngoc Tuyet Minh
protein ubiquitin cua te bao. S tng tac cua E6 oi vi p53 c xem la nguyen
nhan chnh cua bat on nhiem sac the trong te bao xam nhiem bi HPV gay ung th.
1.7.5.6. Protein E7
Protein E7 gom 98 amino acid la mot phosphoprotein lien ket kem co hai vung
cys-X-X-Cys. Cau truc nay cho thay s tng ong vi protein E6, chng to chung
co lien he ve mat tien hoa. Phan au N cua protein E7 cha hai vung tng ng mot
phan vi vung bao ton 1 (CR-1) va hoan toan tng ng vi vung bao ton 2 (CR-2)
cua protein adenovirus E1A va vi mot vung tng t tren khang nguyen T ln cua
SV40. Ca hai vung tng ng tren protein E7 (cd1 va cd 2) gop phan tao nen tiem
nang gay ung th cua no.
Tng t vi E1A va khang the T cua SV40, protein E7 cua HPV tao phc hp
vi protein retinoblastoma (pRB). cac type HPV co kha nang gay ung th (nguy
c cao) ai lc lien ket cua E7 vi pRB ln hn khoang 10 lan so vi cac type HPV
thong thng (nguy c thap) do khac nhau mot amino acid v tr th 21. Ai lc cua
E7 vi pRB co anh hng ln en hieu qua chuyen hoa te bao cua virut. Ngoai ra,
s lien ket E7 vi pRB con lam giai phong mot so nhan to te bao nh nhan to phien
ma E2F. S tang nong o E2F t do trong te bao co the dan en s keo dai tuoi tho
te bao.
1.7.5.7. Protein p53

Protein p53 la mot protein ieu hoa phien ma, co kha nang gan vi mot so trnh
t DNA bang au N, va tao dimer hoac tetramer bang au C. Chc nang cua p53
trong chu trnh te bao la hoat ong iem kiem soat R e chi phoi s tiep dien cua
chu trnh phan bao. Ngoai ra p53 con tang cng kiem soat e a te bao vao s
chet theo chng trnh (apoptosis) khi co sai hong. Protein E6 cua cac type HPV gay
ung th co the tao thanh phc hp vi p53, qua trung gian cua protein ket hp E6
(E6AP). Phc hp E6/p53 gay ra s giam nong o p53 trong te bao do o khien cho
te bao co DNA sai hong co the ton tai qua qua trnh phan bao. Protein cua cac type
HPV nguy c thap (low risk) khong co kha nang gan vi p53 va khong anh hng g
len s on nh p53 trong thc nghiem.
Ta
41 Ngoc Tuyet Minh
1.7.5.8. Retinoblastoma Protein (pRb)

ay la mot phosphoprotein co trong lng phan t khoang 105kD, c
phosphoryl hoa giai oan G2 cua chu trnh te bao. Khi khong c phosphoryl hoa
pRb tao phc vi protein E2F va c che phien ma bang cach gan protein c che. Khi
c phosphoryl hoa, pRb khong con tao phc vi E2F c na, khi o. E2F dimer
hoa vi protein DP va hoat hoa s bieu hien cua mot so gen ieu hoa a te bao
vao giai oan S, nh DNA polymerase , thymidylate synthase, ribonucleotide
redutase, cyclin E va dihydrofolate reductase.
S tang cng E2F t do dan en tang cng chu trnh te bao. Nh a e cap
tren, protein E7 cua HPV nguy c cao co kha nang tao phc lien ket vi pRB va co
ai lc vi pRB cao hn protein E7 nguy c gay lieu thap nhieu lan. Khi protein E7
tao phc vi pRB, pRB khong the tao phc vi E2 va do o E2F co the lam chat
hoat hoa phien ma dan en s tang sinh te bao tao ra khoi u.
1.7.5.9. Protein p27kip I

Thuoc ho Cip/Kip thuoc nhom CKI, p27kip I la mot phosphoprotein nhan co 198
amino acid, co gan CDK au N e gan va c che cyclin kinase D, E, A, B. au
3C cua protein E7 cua HPV co kha nang lien ket vi p27kip I va lam mat hoat tnh
cua no. Nh a e cap, nhom CKI c che hoat ong xuc tien phan bao cua nhan to
CDK, do o khi E7 lien ket vi p27kip I lam cho mat hoat tnh c che, khien cho qua
trnh tien en phan bao xay ra de dang hn.
Ta
42 Ngoc Tuyet Minh
Hnh 6: S nhiem HPV
Hnh 7: Ty le nhiem cac type HPV trong ung th co t cung

1.7.6. Phan loai HPV
Tai hoi ngh thng nien ve Papillomavirus nam 1995 (Quebec, Canada), ngi
ta thong nhat phng phap phan loai cac type HPV da tren s so sanh mc o khac
nhau cua bo gen, cu the la neu co s khac biet hn 10% tren khung oc m cua L1
th c coi nh thuoc mot kieu virut mi. c tnh hn 77 type HPV a c xac
nh va giai trnh t hoan toan. Khoang hn 39 kieu khac ang c giai trnh t
mot phan cho thay tong so cac type HPV len en hn 100.
Da tren cau tao cua nucleic acid, ngi ta chia HPV ra thanh nhieu nhom nho.
Mot trong nhng nhom nho c biet en nhieu nhat la nhom cac HPV xam nhiem
chu yeu be mat niem mac, thng thay nhat ng sinh duc. Hn 40 type HPV
c xac nh thuoc nhom nay trong o c biet nhat la HPV 16. Mot nhom nho
Ta
43 Ngoc Tuyet Minh
khac la nhng virut ai dien bi cac virut tm thay trong cac mun coc loan san dang
bieu mo (epidermodysplasia veruciformis lesions), thng gap nhat la HPV 5.
Nhng virut nay cung c tm thay trong cac vet l cua ngi b suy yeu mien
dch.
1.7.7. Lch s nuoi cay te bao bieu mo co t cung

Nam 1975 Rheinwald va Green a th nghiem nuoi cay hang loat lp te bao bao
qui au sng hoa cua tre s sinh nhng khong thanh cong. Vao nam 1976 Wilbank
va Find lan au tien th nghiem nuoi cay mot mau te bao bieu mo co t cung th
thay te bao co kha nang phat trien in vitro. Nhng ty le thanh cong trong cac loat
th nghiem nuoi cay nay rat han che. Thng lan nuoi cay s cap ty le bam lp
te bao sng hoa rat thap va nhanh chong suy yeu lan nuoi cay th cap. nhng
th nghiem nuoi cay nay nguyen bao si thng chiem u the.
Thi gian sau o cac nha nghien cu bat au chnh thc tien hanh nuoi cay te
bao ung th sng hoa t cac benh nhan b ton thng noi bieu mo co t cung. Mac
du dong te bao ung th co t cung uc Gay va cong s thiet lap t nhng nam 1952
va c s dung rong rai trong cac nghien cu sinh hoc, nhng cha co chng thc
nao xac nhan dong te bao Hela nay co nguon goc t ton thng noi bieu mo. Mai
en khi dong te bao W12 (Sanley va cong s thiet lap vao nam 1989) va CIN 612
(Badell va cong s thiet lap vao nam 1991) ca hai eu co nguon goc t ton thng
bieu mo t cung o thap.
Nhng han che gap trong nuoi cay te bao bieu mo bnh thng cung xay ra
qua trnh nuoi cay te bao ung th co t cung sng hoa, them vao o la s phc tap
cua cac mau sinh thiet co nguon goc khac nhau. Nhng th nghiem sau o cua
Stanley va Parkison cung chng minh rang te bao co t cung sng hoa co the nuoi
cay lien tuc. Ham va cong s th nghiem cac phng phap nuoi cay e xac nh moi
trng dinh dng va phat trien cua cac te bao sng hoa. Ket qua la te bao co t
cung sng hoa co the phat trien moi trng MCDB 153 (Boyce va Ham) va te bao
sng hoa phat trien thanh lp n ma khong trai qua qua trnh biet hoa in vitro.
Tr ngai ln thng gap trong nuoi cay te bao ung th co t cung la s nhiem
khuan, nhiem mycoplasma va nhiem nguyen bao si. V vay moi trng phat trien
Ta
44 Ngoc Tuyet Minh
te bao ung th co t cung ngay t au phai co thuoc khang sinh. Ngoai ra ngi ta
co the kiem tra mot cach co hieu qua s lay nhiem nguyen bao si bang cach thc
hien cac thao tac thanh thuc e loai bo cac dang te bao khac trong qua trnh nuoi
cay. Noi chung, ty le thanh cong khi nuoi cay dong te bao ung th co t cung thng
thap, ty le thanh cong la 1/10 mau sinh thiet.
1.7.8. Th trng dong te bao ung th co t cung

Hien nay phan ln dong te bao ung th co t cung c cung cap bi ATCC
(American Tissue Culture Collection) va DSMZ (Deutsche Sammlung Von
Mikroorganismen und Zellkulturen). Mot so dong c s dung pho bien cac
phong th nghiem hien nay nh: C-33A, C-41, C41 II, Caski, Hela, Hs 588.T, Hs
636.T, HT-3, KB-3-1, ME 180, MS751, SiHa, SISO, SW756, Hela P3, Hela AG,
Hela S3, SNG-N, SNG-PHela S3 (co nguon goc t te bao ung th bieu mo co t
cung ngi) nuoi cay trong moi trng HAMs F12 va 10% FBS va thch hp phat
trien dong trong moi trng N16 HHF. Hela S3 do T.T va P.I.Drarcus thiet lap t
nam 1955.
Dong te bao Hela S3 cung c s dung e nghien cu cac khang nguyen co
nguon goc t cac virut sinh u. Cac nghien cu cho thay rang cac khang nguyen co
nguon goc t cac virut sinh u co the c nhan biet bi cac te bao T. V du ien
hnh nhat ngi la cac peptid co nguon goc t protein E7 cua HPV 16, virut nay co
mat trong nhieu ung th bieu mo co t cung, cac te bao T au oc te bao co the tan
cong va giet cac te bao ung th ngay khi chung xuat hien.
Dong te bao SNG-M va SNG-P la khoi u di can co nguon goc t ung th co t
cung ngi c nuoi cay trong moi trng HAMs F12 vi 20% FBS (Ishiwatari.Y
va Nozawas). ay la dong te bao phat trien kha tot nuoi cay hn 13 thang va cay
chuyen hn 65 lan va van tiep tuc phat trien on nh. SNG-M va SNG-P c s
dung tren c s phan tch chu trnh te bao e nghien cu so sanh gia mot te bao
bnh thng va te bao ung th co t cung ang c xa tr e xem xet viec xa tr vao
thi iem nao la at hieu qua nhat. Trong moi trng nuoi cay phong th nghiem,
hau het cac te bao bnh thng ch phan chia trong vong 20 50 the he th ngng
lai. Chung tiep tuc phan chia cho en khi chung cham phai nhau th ngng lai khong
Ta
45 Ngoc Tuyet Minh
phan chia na, hien tng nay c goi la c che phu thuoc mat o. Ngc lai cac
te bao ung th khong phan ng vi c che kiem soat nay.
Bang cach nghien cu cac te bao ung th trong moi trng nuoi cay cac nha
nghien cu thay rang te bao ung th khong chu anh hng g hoac chu anh hng
cac nhan to kiem soat phan bao kem hn cac te bao bnh thng. Cac te bao ung th
co the phan chia mai khong b gii han chng nao chung con c cung cap chat
dinh dng. Neu te bao ung th ngng phan chia chung se ngng tai nhng thi
iem ngau nhien hn la ngng ung thi iem khung hoang. Viec x ly ung th
bang phng phap xa tr la nham ngan chan e cac te bao ung th ngng phan chia.
V vay khi te bao b huy diet trong gian ky th moi hoat ong phan bao se ngng
ngay. Hien tng nay co the thay khi chieu xa lieu cao.
Hela P3 (co nguon goc t te bao ung th bieu mo co t cung ngi) c nuoi
cay trong moi trng DM210 khong huyet thanh. Dong SiHa va CaSki (cung co
nguon goc t te bao ung th bieu mo co t cung ngi) c nuoi cay trong moi
trng DMEM vi 10% fetal cafl serum co s dung them 0,25% trypsin khi tao
dong. Dong SiHa va CaSki co kha nang kiem soat HPV16 va Hela P3 co the khang
lai HPV18.
Cac nha nghien cu a s dung nhieu dong te bao nuoi cay nh Hela P3, CaSki,
SiHa e th nghiem viec ieu tr ung th co t cung bang cach da tren s mat tac
dung tng ho gia protein vi protein, co lien quan en protein gay c che ung th.
Chang han nh oncoprotein E7 va E6 lien ket, tac dung tng ho nay vi protein
virut nh virut papilloma ngi co the giai phong ra nhng protein c che ung th
va ieu nay co the co ch trong ieu tr ung th co t cung. Bang cac nghien cu
nuoi cay te bao cac nha khoa hoc con chng minh rang s thay oi tien trnh cua
nhiem sac the va s xam nhap cua HPV lam phat trien ung th co t cung. Ngoai
cac HPV ang chu y nh HPV 16, HPV 18 con co cac HPV khac nh HPV31,
HPV33, HPV35, HPV45 c tm thay trong cac khoi u co t cung. Cac nha khoa
hoc cho rang s xuat hien cua cac HPV nay co lien quan en van e a ly (HPV 45
tm thay pho bien cac benh nhan n Chau Phi va cac nc Nam My).
Trong nhieu nam qua bang cac th nghiem nuoi cay t dong te bao Hela, CaSki
cac nhom nghien cu a ch ra c nhieu gen c che ung th, trong o co gen
BRCA 1 khu tru 17q bieu hien benh ung th vu va ung th t cung. Cac nha khoa
Ta
46 Ngoc Tuyet Minh
hoc cho rang neu s ieu tr da tren gen c che ung th c phat trien th cac nha
ung th hoc co kha nang xay dng nhng test chan oan thch hp va benh nhan
hoan toan c li t bien phap ieu tr nh the. S mat thng xuyen cac gen c
che ung th at ra cho cac nha khoa hoc mot cau hoi co phai benh nay giong nh
benh khiem khuyet di truyen. Va cau tra li la tm bien phap ieu tr bang cach thay
the gen chnh la muc tieu thch hp nhat. Cong trnh nghien cu nhieu nam qua a
cho thay rang gen p53 co the rat phu hp e dung ngan chan s sinh san te bao ung
th hoac e pha huy cac te bao nay.
Ta
47 Ngoc Tuyet Minh
1. VAT LIEU HOA CHAT

1.1.
oi tng nghien cu
e thc hien c nhng th nghiem nghien cu ve e tai nay, chung toi

c s giup va ho tr ve nguon mau va nhng thong tin can thiet cua nguon
mau t Benh vien Ung Bu cung cap. Sau khi tien hanh kham lam sang va chan
oan giai oan benh, cac bac s tai phong kham se lay sinh thiet nguon mau t cho
sui cua co t cung cua benh nhan. Tuy theo tng giai oan, cho sui co the t 2
5cm. Trong lng mau c cung cap thng khoang 1,4 1,6g. Nguon mau co
c t nhieu benh nhan co o tuoi khac nhau, giai oan benh khac nhau, ni sinh
song khac nhau. Qui trnh x ly nguon mau se c tien hanh tai phong th nghiem
Cong nghe Sinh Hoc Phan T Lab C, trng ai hoc Khoa Hoc T Nhien.
Hnh 6: Mau sinh thiet

1.2. Dung cu Thiet b Hoa chat
I.2.1. Dung cu
-
a petri (ng knh 10cm)

Erlen 50ml, 100ml, 250ml
en con
Becher 50ml, 100ml, 250ml
Micropipette (10-100ml va 100-1000ml)
Kep cong va thang
Keo cong va thang
au type 10-100ml va 100-1000ml
Buong em NEUBAUER
Ta
48 Ngoc Tuyet Minh
Lamelle
Ong ly tam
Bnh roux
Eppendorf 1500l
Bnh xt con
1.2.2. Thiet b
Tu cay vo trung
Tu am CO2 dang kho
Noi ap suat (autoclave)
May khuay t gia nhiet
May ly tam
May anh ky thuat so
Tu say
Tu lanh
Knh hien vi ien t
Knh hien vi soi ngc
Microfilter
Can ien t
1.2.3. Hoa chat

Dung dch PBSA
g/l
NaCl
8,00
KCl
0,20
2,16
Na2HPO4.7H2O
0,20
KH2PO4
1000ml
H2O cat va u
pH
7,2
Bo sung 50g/ml Gentamicin, 5g/ml Ampotericin B sau khi PBS a c hap kh
trung.
- Trypsin 1%
Trypsin
1g
PBS vo trung (vt)
100ml
Loc vo trung bang
0,45 0,22m
Microfilter
Bao quan lanh 4oC.
- EDTA 1%
EDTA
1g
Nc cat
100ml
Hap kh trung
Ta
49 Ngoc Tuyet Minh
Trypsin EDTA
Trypsin 0,25% vt
25ml
EDTA 1% vt
500m
PBS vt
24ml
o
Bao quan lanh 4 C.
Pen Strep 200X
Penicilin G
1.000.000 n v
Streptomycin
1g
sulfate
Loc vo trung bang microfilter 0,45 0,22m
Bao quan lanh 20oC
Gentamicin
Gentamycin
80mg/2ml
Bao quan lanh 20oC.
Ampotericin B
Ampotericin B
50mg
Nc cat
10ml
Bao quan lanh 4oC.
Moi trng DMEM (Dulbeccos modification of Eagles medium) vi huyet
thanh 10%.
L-Arginine HCl
84 mg/l
L-cystine
48
L-glutamic acid
75
L-glutamine
584
Glycine
30
42
L-histidine.HCl.H2O
L-isoleucine
105
L-leucine
105
L-lysine.HCl
146
L-methionine
30
L-phenylalanine
66
L-serine
42
L-threonine
95
L-tryptophan
16
L-tyrosine
72
L-valine
94
200
CaCl2
0,10
Fe(NO3)3.9H2O
KCl
400
Ta
50 Ngoc Tuyet Minh
MgSO4.7H2O
200
NaCl
6400
3700
NaHCO3
125
Na2HPO4.H2O
D-Ca pantothenate
4
Choline chloride
4
Folic acid
4
i-insitol
7,2
Nicotinamide
4
Pyridoxal HCl
4
Riboflavin
0,4
Thiamine HCl
4
D-Glucose
4500
Phenol red
15
Sodium pyruvate
110
Bo sung huyet thanh FBS (Fetal Bovine Serum) 10%
2. PHNG PHAP NGHIEN CU E TACH TE BAO UNG TH

2.1. Cac yeu to can c khao sat
-
Tach te bao ung th co t cung bang phng phap c hoc.

- Tach te bao ung th co t cung bang quy trnh Trypsin am.
- Khao sat thi gian khuay manh mo.
- Th nghiem nuoi cay s cap te bao ung th co t cung.
2.2. Trnh t th nghiem
Bc 1: Thu nhan mau va ghi nhan cac thong tin lien quan en mau vat, bao
gom:
- Ten tuoi ngi cho mau
- Chan oan giai oan benh
- Thi gian lay mau (ngay, gi)
- Thi gian tien hanh thao tac tai phong th nghiem
Bc 2: Tien hanh chieu UV kh trung tu cay, mau, dung cu va hoa chat (hap
kh trung neu can)
Bc 3: Tien hanh tach te bao t mau mo bang phng phap c hoc hoac bang
quy trnh Trypsin am.
Bc 4: Th lai quy trnh tach te bao bang cach thay oi nong o Trypsin ghi
nhan mat o te bao theo thi gian.
Bc 5: Th nghiem nuoi cay s cap te bao.
Ta
51 Ngoc Tuyet Minh
2.3. Cac quy trnh chnh s dung trong thc nghiem.

2.3.1. Quy trnh vo trung
2.3.1.1. Dung cu Hoa chat
-
a) Dung cu
Dung cu bang thuy tinh va kim loai c vo trung bang cach hap kh trung hi
nc vi autoclave (121oC 1 atm trong 30 phut).
Dung cu nha: lau bang con 70o, e kho t nhien va chieu UV 30 phut trc khi
s dung.
b) Hoa chat
PBS, nc cat: hap kh trung hi nc
Moi trng DMEM, enzym Trypsin: loc vo trung.
c) Thao tac loc vo trung
Dung mot ong tiem mi (khong can kim tiem) hoac ong hut lay dung dch can
loc.
M bao b filter, tra au ong tiem vao au filter, nhac filter ra khoi bao b.
Ep Piston hoac bao cao su e dch loc chay vao mot bnh cha vo trung.
at mieng loc tr lai bao b, rut ong tiem ra, tiep tuc hut dung dch e loc neu
can thiet.
2.3.2. Quy trnh x ly mau mo tai phong kham benh vien Ung Bu.
Ta
52 Ngoc Tuyet Minh
Chan oan giai oan benh bang mo vt
Bam mau sinh thiet
Ghi nhan thong tin mau, thi gian thu mau
Ra mau qua dung dch PBSA co khang sinh

Penicillin Streptomycin trong 10 phut
Ra mau qua dung dch PBSA co khang sinh

Gentamycin Ampotericin B trong 10 phut
Tr mau trong eppendorf cha san 1ml moi

trng DMEM co huyet thanh FBS 10%
Tr eppendorf nhiet o 4oC
Chu y:
Nhng thao tac tren c tien hanh trong ieu kien khong vo trung, v vay de dang
xay ra s nhiem vi sinh vat vao mau vat. e tranh c s nhiem th cac dung cu va
dung dch phai c vo trung, thao tac x ly mau phai nhanh va c thc hien gan
en con. Viec s dung khang sinh trong dung dch PBSA e ra mau nham muc ch
co the loai bo hoac giam s nhiem vi sinh vat vao mau vat.
2.3.2. Quy trnh tach te bao bang quy trnh trypsin am.
(a) Chuyen mau c tr trong eppendorf co cha moi trng DMEM 10% FBS vao
a petri (ng knh10cm).
(b) Ra mau bang PBSA co khang sinh Gentamycin Ampotericin B t 2 3 lan e
lam sach mau.
(c) S dung keo cong hoac thang cat mau mo ra thanh tng manh nho, tiep tuc cat
nhuyen cac manh mo.
Ta
53 Ngoc Tuyet Minh
(d) Chuyen nhng manh mo a cat nho vao erlen 50ml, them vao 5ml TrypsinEDTA.
(e) Khuay t nhiet o 37oC t 15 60 phut, khao sat mat o te bao theo thi gian.
(f) Sau 15 phut lay erlen ra khoi may khuay t, e yen trong vong 2 -3 phut. Hut
phan dung dch noi pha tren cho vao ong ly tam, em ly tam 1000v/ 5 phut.
(g) Phan mau con lai trong erlen tiep tuc khuay 37oC. Sau 15 phut se tien hanh
thao tac nh phan (f) cho en khi nao thu c toan bo lng te bao can thiet.
(h) Sau khi ly tam 1000v/ 5 phut, loai bo phan noi, sau o them vao 5ml moi trng
DMEM 10% FBS.
(i) em mat o te bao ung th (tr te bao mau, hong cau, bach cau ).
(j) Cho mau vao gieng nuoi trong tu am 37oC, 5% CO2.
(k) Quan sat ket qua sau 24 gi, 48 gi, 76 gi
2.3.3. Quy trnh tach te bao bang phng phap c hoc.
(a) Chuyen mau c tr trong eppendorf co cha moi trng DMEM 10% FBS vao
a petri (ng knh10cm).
(b) Ra mau bang PBSA co khang sinh Gentamycin Ampotericin B t 2 3 lan e
lam sach mau.
(c) S dung keo cong hoac thang cat mau mo ra thanh tng manh nho, tiep tuc cat
nhuyen cac manh mo.
(d) Them vao a petri 1ml FBS, chuyen nhng manh mo vao bnh roux.
(e) e yen trong vong 24 gi, e giup cho s bam te bao c xay ra.
(f) Them vao 5ml moi trng DMEM, cho vao tu am 37oC, 5% CO2.
(g) Quan sat ket qua sau 24 gi, 48 gi,
Ta
54 Ngoc Tuyet Minh
Hnh 15: Mau sinh thiet c tr trong eppendorf cha 1ml moi trng
DMEM co huyet thanh FBS 10%
Hnh 16: Chuyen mau c tr trong eppendorf co cha moi trng DMEM
10% FBS vao a petri
Hnh 17: S dung keo cong hoac thang cat nhuyen mau mo
Khuay t nhiet o 37oC t 15
60 phut, khao sat mat o te
bao theo thi gian
Them vao a petri 1ml FBS, chuyen

nhng manh mo vao bnh roux
Hnh 18: Hut mau sau khi khuay cho vao

ong ly tam
Ta
55 Ngoc Tuyet Minh
Hnh 19: Chuyen nhng manh mo

vao bnh roux
Hnh 20: em ly tam 1000v/ 5 phut
Hnh 21: Cho bnh roux vao

tu am 37oC, 5% CO2
Hnh 22: Cho mau vao gieng nuoi at

trong tu am 37oC, 5% CO2
2.3.4. Phng phap xac nh mat o te bao bang phong em NEUBAUER

-
Ra phong em bang nc cat

Say kho
Ra lai phong em bang con 70oC
Say kho
at lamelle len phong em
ong bt chan sang
Xac nh th trng phong em di knh hien vi quang hoc
Cho mau vao phong em
Xac nh mat o te bao theo quy tac em pha tren, ben phai cua cac o trong
phong em
Phong em gom 9 o ln
Moi o ln gom 16 o nho
em so te bao trong 5 o ln (Vung 1,2,3,4,5)
Moi o ln co the tch: 1 x 1 x 0,1 = 0,1 mm3 = 10-4 ml
Mat o te bao = (so te bao cua 5 o ln / 5) x 104 (so te bao/ ml)
Ta
56 Ngoc Tuyet Minh
Hnh 23: Cac v tr xac nh so te bao tren phong em
1. KET QUA KHAO SAT KHA NANG TACH TE BAO UNG TH CO T

CUNG BANG MAY LAC BA NONG O TRYPSIN KHAC NHAU
Bang 2: Thong tin ve nguon mau
Chan oan giai oan

Tuoi ngi cho mau
Thi iem phau thuat
Thi iem tien hanh
th nghiem
Mau 1
IIA
77
8 gi 5 phut
13 gi 30 phut
Mau 2
IIA
58
8 gi 30 phut
13gi 30 phut
Mau 3
IIB
60
9 gi 5 phut
13 gi 30 phut
Bang 3: Mat o te bao ung th co t cung tai cac thi iem khuay
Thi gian
khuay (phut)
30
60
Ta
57 Ngoc Tuyet Minh
Mat o te bao (STBx104/ml)

Mau 1 khuay vi
Mau 2 khuay vi
Mau 3 khuay vi
Trypsin 0,125%
Trypsin 0,25%
Trypsin 0,5%
6
11
15
10
21
35
90
120
15
26
28
37
59
60
Mat o te bao (STBx104)
47
59
47
50
35
37
40
28
26
30
60 phu t
21
20
15
11
10
10
30 phu t
15
90 phu t
120 phu t
0
Ma u 1
Ma u 2
Ma u 3
Bieu o 1: Mat o te bao ung th co t cung thu c theo thi gian lac
Bang 2 va bieu o 1 cho thay:

-
Khi ta cat mau mo thanh nhng manh nho, khao sat mat o te bao thu c bang
cach s dung may lac vi 3 nong o trypsin khac nhau (0,125%, 0,25%, 0,5%).
Vi ba nong o tren, sau khi lac 120 phut mat o te bao thu c cha en mc
cao nhat. ieu nay cho thay rang viec s dung may lac tac ong en s tach ri
cac te bao phai ton mot khoang thi gian kha dai, se anh hng en thi gian th
nghiem.
Do o chung toi a tien hanh khao sat cung vi ba nong o trypsin tren bang
phng phap khuay t nhiet o 37oC.

CUNG BANG MAY KHUAY T 37OC VI BA NONG O TRYPSIN KHAC
NHAU
Chan oan giai oan
Tuoi ngi cho mau
Thi iem phau thuat
Ta
58 Ngoc Tuyet Minh
Mau 1
IB
57
8 gi 15 phut
Mau 2
IIA
68
8 gi 20 phut
Mau 3
IIB
48
8 gi 30 phut
Thi iem tien hanh

th nghiem
13 gi 5 phut
13gi 5 phut
13 gi 5 phut
Thi gian khuay
(phut)
30
60

Mau 1 khuay vi
Mau 2 khuay vi
Mau 3 khuay
Trypsin 0,125%
Trypsin 0,25%
vi Trypsin
0,5%
13
27
35
22
30
82
90
120
27
30
60
47
82
90
67
43
80
67
70
60
60
47
50
40
30
20
22
27 30
27 30
43
35
30 phu t
60 phu t
90 phu t
120 phu t
13
10
0
Ma u 1
Ma u 2
Ma u 3
Bieu o 2: Mat o te bao ung th co t cung thu c theo thi gian khuay
-
Mau 1: khi khuay vi trypsin 0,125%, sau 120 phut khuay mat o te bao khong
at en mc cao nhat, ieu nay se anh hng en thi gian th nghiem.
Mau 2: khi khuay vi trypsin 0,25%, sau 90 phut khuay mat o te bao at en
mc cao nhat. nong o trypsin 0,25%, ty le te bao song la 98%.
Mau 3: khi khuay vi trypsin 0,5%, sau 60 phut mat o te bao at en mc cao
nhat, ty le te bao song la 85%.
Qua ket qua tren chung toi chon nong o trypsin 0,25%. Mac du thi gian khuay e
at c mat o te bao la 90 phut nhng ty le te bao song cao.
Ta
59 Ngoc Tuyet Minh

CUNG NONG O TRYPSIN 0,25%
Mau 1
Mau 2
Chan oan giai oan
IIB
IIA
Tuoi ngi cho mau
55
56
Thi iem phau
8 gi 15 phut
8 gi 30 phut
thuat
Thi iem tien hanh
13 gi 30 phut
13gi 30 phut
th nghiem
Mau 3
IIB
76
9 gi 10 phut
13 gi 30 phut
Thi gian
khuay (phut)
Mau 1
Mau 2
Mau 3
30
13
95
15
60
24
113
35
90
120
37
23
168
137
180
59
46
168
160
137
140
113
120
30 phu t
95
100
60 phu t
80
59
60
37
40
20
24
13
35
23
46
90 phu t
120 phu t
15
0
Ma u 1
Ma u 2
Ma u 3
-
Khi khuay vi nong o trypsin 0,25% ba mau 1, 2, 3, sau 90 phut mat o te bao
eu at en mc cao nhat, ty le te bao song la 95% - 98%.
Ta
60 Ngoc Tuyet Minh
Chung toi a khac phuc khuyet iem ve thi gian khuay bang cach tien hanh th
nghiem khuay vi nong o trypsin 0,25%, va them vao EDTA (chat nen giup
cho tac ong trypsin len s tach cac te bao co hieu qua va nhanh hn) vi ty le
EDTA:Trypsin la 1:4.

CUNG BANG PHNG PHAP KHUAY T GIA NHIET VI NONG O
TRYPSIN 0,25% + EDTA 0,02%.
Chan oan giai oan
Tuoi ngi cho mau
Thi iem phau thuat
Thi iem tien hanh th
nghiem
Mau 1
IIA
41
8 gi 50 phut
14 gi 00 phut
Mau 2
IB
55
9 gi 00 phut
14 gi 00 phut
Mau 3
IB
44
9 gi 15 phut
14 gi 00 phut
Thi gian
khuay (phut)
Mau 1
Mau 2
Mau 3
15
28
11
13
30
38
23
25
45
60
90
Ta
61 Ngoc Tuyet Minh
48
64
54
33
36
27
29
35
31
70
64
60
54
48
50
30
15 phu t
38
40
33
28
23
20
36
35
27
25
29
31
45 phu t
60 phu t
13
11
30 phu t
90 phu t
10
0
Ma u 1
Ma u 2
Ma u 3
Bieu o 4: Mat o te bao ung th co t cung thu c theo thi gian

khuay
Chan oan giai oan

Tuoi ngi cho mau
Thi iem phau thuat
Thi iem tien hanh
th nghiem
Mau 4
IIB
51
8 gi 30 phut
14 gi 00 phut
Mau 5
IIB
52
9 gi 15 phut
14 gi 00 phut
Mau 6
IB
50
9 gi 30 phut
14 gi 00 phut
Thi gian
khuay (phut)
15
30
45
60
90
Ta
62 Ngoc Tuyet Minh
Mau 4
122
153

Mau 5
66
92
Mau 6
14
16
180
235
201
142
188
145
23
36
32
250
235
201
200
188
180
153
150
142
15 phu t
145
30 phu t
122
45 phu t
92
100
60 phu t
66
50
14 16
23
36 32
90 phu t
0
Ma u 4
Ma u 5
Ma u 6
Bieu o 5: Mat o te bao ung th co t cung thu c theo thi gian

khuay
Chan oan giai oan

Tuoi ngi cho mau
Thi iem phau thuat
Thi iem tien hanh
th nghiem
Mau 7
IIA
38
8 gi 45 phut
14 gi 00 phut
Mau 8
IIB
56
9 gi 25 phut
14 gi 00 phut
Mau 9
IIA
43
9 gi 35 phut
14 gi 00 phut
Thi gian
khuay (phut)
15
30
45
60
90
Ta
63 Ngoc Tuyet Minh
Mau 7
38
55

Mau 8
35
68
Mau 9
25
43
69
86
67
85
104
92
68
78
59
120
104
100
80
69
85
78
67
15 phu t
68
68
59
55
60
40
92
86
43
38
35
30 phu t
45 phu t
60 phu t
25
90 phu t
20
0
Ma u 7
Ma u 8
Ma u 9

Mau 10
IIA
56
10 gi 10
phut
14 gi 30
phut
Chan oan giai oan

Tuoi ngi cho mau
Thi iem phau thuat
Thi iem tien hanh
th nghiem
Mau 11
IB
45
9 gi 30 phut
Mau 12
IIB
57
9 gi 45 phut
14 gi 30 phut
14 gi 30 phut
Thi gian
khuay (phut)
15
30
45
60
90
Ta
64 Ngoc Tuyet Minh

Mau 10
Mau 11
Mau 12
37
13
50
42
26
72
53
69
49
35
41
29
88
125
97
140
125
120
97
100
88
80
60
40
72
69
37
42
53
49
26
35
30 phu t
45 phu t
50
41
15 phu t
60 phu t
29
90 phu t
13
20
0
Ma u 10
Ma u 11
Ma u 12
Chan oan giai oan

Tuoi ngi cho mau
Thi iem phau
thuat
Thi iem tien hanh
th nghiem
Mau 13
IIA
49
8 gi 15 phut
Mau 14
IIB
49
9 gi 30 phut
Mau 15
IIB
69
8 gi 45 phut
13 gi 30 phut
13 gi 30 phut
13 gi 30 phut
Thi gian
khuay
(phut)
15
30
45
60
90
Ta
65 Ngoc Tuyet Minh
Mau 13

Mau 14
Mau 15
27
35
78
92
62
86
59
79
62
123
158
119
99
120
102
158
160
140
123
120
99
92
100
27
15 phu t
30 phu t
45 phu t
62
62
59
60
102
86
78
79
80
40
120
119
60 phu t
35
90 phu t
20
0
Ma u 13
Ma u 14
Ma u 15
Chan oan giai oan

Tuoi ngi cho mau
Thi iem phau
thuat
Thi iem tien hanh
th nghiem
Mau 16
IB
49
9 gi 15 phut
Mau 17
IIB
49
9 gi 30 phut
Mau 18
IIA
69
9 gi 40 phut
13 gi 30 phut
13 gi 30 phut
13 gi 30 phut
Thi gian
khuay (phut)
15
30
45
60
90
Ta
66 Ngoc Tuyet Minh
Mau 16
17
22

Mau 17
33
56
Mau 18
20
42
23
39
29
75
91
79
58
72
67
100
91
90
75
80
79
72
70
50
20
42
39
40
30
58
56
60
17
29
22 23
67
33
15 phut
30 phut
45 phut
60 phut
20
90 phut
10
0
Ma u 16
Ma u 17
Ma u 18
Chan oan giai oan

Tuoi ngi cho mau
Thi iem phau thuat
Thi iem tien hanh
th nghiem
Mau 19
IIA
60
8 gi 15 phut
13 gi 30 phut
Mau 20
IIA
41
8 gi 20 phut
13 gi 30 phut
Mau 21
IB
64
8 gi 35 phut
13 gi 30 phut
Thi gian
khuay (phut)
15
30
45
60
90
Ta
67 Ngoc Tuyet Minh
Mau 19
22
38

Mau 20
38
42
Mau 21
12
26
69
89
72
73
98
79
39
40
32
98
100
89
90
79
80
69
70
73
72
15 phu t
60
50
38
40
30
38
30 phu t
42
39 40
32
26
22
20
45 phu t
60 phu t
90 phu t
12
10
0
Ma u 19
Ma u 20
Ma u 21
Bieu o 10: Mat o te bao ung th co t cung thu c theo thi gian khuay.

Chan oan giai oan
Tuoi ngi cho mau
Thi iem phau thuat
nghiem
Mau 22
IIA
70
8 gi 45 phut
14 gi 30
phut
Mau 23
IB
45
9 gi phut
14 gi 30 phut
Mau 24
IB
57
9 gi 10 phut
14 gi 30 phut
Thi gian
khuay
(phut)
15
30
45
60
90
Ta
68 Ngoc Tuyet Minh
Mau 22

Mau 23
Mau 24
39
55
15
28
14
23
79
96
87
35
44
32
28
38
29
96
100
87
90
79
80
70
50
40
15 phu t
55
60
30 phu t
44
39
28
30
35
15
20
45 phu t
38
32
23
28
29
14
60 phu t
90 phu t
10
0
Ma u 22
Ma u 23
Ma u 24

Mau 25
IB
44
8 gi 15 phut
14 gi 30 phut
Chan oan giai oan

Tuoi ngi cho mau
Thi iem phau thuat
nghiem
Mau 26
IB
58
8 gi 30 phut
14 gi 30 phut
Mau 27
IB
56
8 gi 45 phut
14 gi 30
phut
Thi gian
khuay (phut)
15
30
45
60
90
Ta
69 Ngoc Tuyet Minh
Mau 25
12
15

Mau 26
22
28
Mau 27
14
23
28
35
21
32
39
25
26
37
32
39
40
35
32
32
28
28
30
26
25
25
21
20
15
37
35
23
22
15 phu t
30 phu t
15
14
12
10
45 phu t
60 phu t
90 phu t
5
0
Ma u 25
Ma u 26
Ma u 27

Chan oan giai oan
Tuoi ngi cho mau
Thi iem phau thuat
Thi iem tien hanh
th nghiem
Mau 28
IIB
68
8 gi 30 phut
14 gi 30 phut
Mau 29
IIB
49
8 gi 20 phut
14 gi 30 phut
Mau 30
IIA
64
8 gi 45 phut
14 gi 30 phut
Thi gian
khuay (phut)
15
30
45
60
90
Ta
70 Ngoc Tuyet Minh
Mau 28
42
58

Mau 29
38
52
Mau 30
32
46
67
89
72
73
96
79
59
70
62
96
100
89
90
79
80
67
70
73
72
59
58
60
50
42
40
70
52
62
15 phu t
30 phu t
46
45 phu t
38
32
60 phu t
30
90 phu t
20
10
0
Ma u 28
Ma u 29
Ma u 30
Cac ket qua khao sat kha nang tach te bao ung th co t cung bang phng phap
khuay t gia nhiet vi nong o trypsin 0,25% - EDTA 0,02% c trnh bay tren
cho thay:
-
Mat o te bao tang dan trong khoang thi gian khuay t 15 45 phut, va sau khi
khuay 60 phut mat o te bao at mc cao nhat. Neu tiep tuc khuay th mat o te
bao giam dan.
Vi thi gian va mat o thu c nh tren co the thiet lap quy trnh phu hp e
co the thu nhan hoan toan tong so lng te bao. Sau khi khuay 30 phut tien hanh
thu te bao lan mot, tiep tuc e khuay tiep t 15 phut 30 phut cho en khi cac
te bao c tach ri hoan toan, tien hanh thu te bao lan hai. Nh vay vi nong
o trypsin la 0,25% va EDTA 0,02% va thi gian khuay t 15 60 phut, co the
thu c mat o te bao t 1x105 5x106 e tien hanh nuoi cay s cap.
Mot ieu ang chu y la ta thay co s chenh lech mat o te bao thu c sau khi
khuay gia cac benh nhan theo tng giai oan benh.
Sau ay la bang thong ke mat o te bao sau khi khuay theo tng giai oan:
Giai oan IB:

Bang 27: Mat o te bao cua mau benh pham giai oan IB thu c theo thi
gian khuay
Thi gian
khuay
15 phut
Ta
71 Ngoc Tuyet Minh
30 phut
45 phut
60 phut
90 phut
Mau 2
Mau 3
Mau 6
Mau 11
Mau 16
Mau 21
Mau 23
Mau 24
Mau 25
11
13
14
13
17
12
15
14
12
23
25
16
26
22
26
28
23
15
33
29
23
35
23
39
35
28
28
36
35
36
41
39
40
44
38
35
27
31
32
29
29
32
32
29
21
Mau 26
Mau 27
22
14
28
23
32
26
39
37
25
32
Giai oan IIA:

Bang 28: Mat o te bao cua mau benh pham giai oan IIA thu c theo thi
gian khuay
Thi gian
15 phut
30 phut
45 phut
60 phut
90 phut
khuay
Mau 1
28
38
48
54
64
Mau 7
38
55
69
67
86
Mau 9
25
43
68
59
78
Mau 10
37
42
53
49
69
Mau 13
27
35
59
62
79
Mau 18
20
42
58
67
72
Mau 19
22
38
69
72
89
Mau 20
Mau 22
Mau 30
38
39
32
42
55
46
73
79
59
98
96
70
79
87
62
Giai oan IIB:

Bang 29: Mat o te bao cua mau benh pham giai oan IIB thu c theo thi
gian khuay
Thi gian
15 phut
30 phut
45 phut
60 phut
90 phut
khuay
Mau 5
66
92
142
145
188
Mau 8
35
68
85
92
104
Mau12
50
72
88
97
125
Mau 14
78
92
123
119
158
Ta
72 Ngoc Tuyet Minh
Mau 15
Mau 17
Mau 28
Mau 29
62
33
42
38
86
56
58
52
99
75
67
73
120
91
89
96
102
79
72
79
Bien luan:
Trong ieu kien cho phep th nghiem ch co the s dung cac mau sinh thiet cua
benh nhan b ton thng ung th cac giai oan benh IB, IIA, IIB.
-
Khi quan sat cac te bao c tach ri trong buong em tren knh hien vi ien t,
thay rang van con hien dien hong cau, te bao mau, bach cau Da vao ac
iem te bao ung th c e cap tren, va phan biet vi cac te bao khac khong
phai la te bao ung th ta co the em c so lng te bao ung th mot cach
tng oi.
bang 18, 19, va 20 eu cho thay:
cac giai oan benh khac nhau, mat o te bao eu tang dan theo thi gian
khuay t 15 phut 40 phut, va sau khi khuay 60 phut mat o te bao at mc cao
nhat, va neu tiep tuc khuay mat o te bao se giam dan.
Ngoai ra, co s chenh lech mat o te bao gia cac giai oan, giai oan IIA mat
o te bao thu c thap hn IIB, va mat o te bao thu c IB thap hn so vi
IIA va IIB. Do cac bang thong ke tren ch c thc hien tren so lng mau han
che, co the giai thch mot cach tng oi cho s chenh lech tren nh sau:
giai oan IB, a so hien dien nhieu cac te bao viem (bach cau a nhan hay n
nhan), so lng te bao ung th ton tai t va kho phan biet vi te bao khac do hnh
thai, cau truc bat thng cua chung cha bieu hien ro.
giai oan IIA, IIB so lng te bao co hnh thai, cau truc bat thng hien dien
nhieu hn, tnh a dien cua te bao the hien ro hn va giup phan biet ro hn vi
cac te bao khac.
Nhan xet:
-
Qua qua trnh tien hanh th nghiem va cac ket qua thu c, co the rut ra ket
luan rang khi khuay mau nhiet o 37oC vi dung dch Trypsin 0,25% - EDTA
0,02% se thu c mat o te bao nh mong muon (1x105 5x106).
Ta
73 Ngoc Tuyet Minh
Tuy nhien, cac ieu kien lay nguon mau, nhng sai sot trong qua trnh tien hanh
th nghiem a mot phan nao o anh hng en ket qua th nghiem.
TAI LIEU THAM KHAO

TAI LIEU A C XUAT BAN
1. Nguyen Chan Hung (2004), Ung bu hoc noi khoa, NXB Tp.Ho Ch
Minh.
2. Le nh Oanh (2001), Benh hoc cac khoi u, NXB Ha Noi.
3. Phan Kim Ngoc (2002), Giao trnh thc tap c s Cong nghe sinh hoc
ong vat, NXB ai hoc quoc gia Tp.Ho Ch Minh.
4. T ien bach khoa sinh hoc (2003), NXB Khoa hoc Ky thuat.
5. Simon P.Langdon (2003), Cancer cell culture, Human Press.
6. George H. Fried (1999), Theory and problem biology, Mc Graw Hill.
TAI LIEU TRUY CAP QUA INTERNET
7.
Kwasniewska A, Semczuk M, Sikorski M and Kraczkowski J:Use of the

Digene Hybrid Capture System for identification of the human
papillomavirus (HPV-human papillomavirus) in women with cervical
dysplasia. Przegl Lek 54: 848-850, 1997.
8. Ho GY, Bierman R, Beardsley L, Chang CJ and Burk RD: Natural history of

cervicovaginal papillomavirus infection in young women. N Engl J Med
338: 423-428, 1998.
9. Saito J, Hoshiai H and Noda K: Type of human papillomavirus and
expression of p53 in elderly women with cervical cancer. Gynecol Obstet
Invest 49: 190-193, 2000.
10. Stanley MA (1986): Human cervical epithelium. In webber MM, Sekely LI.
11. Stanley MA, Parkinson EK (1979): Growth requirements of human cervical
epithelial cells in culture.Int J Cancer 24.
Ta
74 Ngoc Tuyet Minh
12. http//www.google.com.vn/search?q=cache:Yvv6UEJ+method+
identification +cervical+carcinoma+cell.
13. http//www.google.com.vn/search?q= cache +hela.
14. http//www.pubmedcentral.nih/gov/article/rapid+induction+of+senescence+in
+human+cervical+carcinoma+cell.
15. http//www.pnas.org/cgi/dol/10.1073=cell+culture+forensics.
Ta
75 Ngoc Tuyet Minh

Tuyet Minh

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Th nghiem phan lap va nuoi cay te bao ung th co t cung

Th nghiem phan lap va nuoi cay te bao ung th co t cung

1.1. MOT SO MOC LCH S NGHIEN CU VE UNG TH

Th nghiem phan lap va nuoi cay te bao ung th co t cung

1.2. NHNG AC TNH CUA UNG TH NGI

Th nghiem phan lap va nuoi cay te bao ung th co t cung

Th nghiem phan lap va nuoi cay te bao ung th co t cung

1.2.2. Sinh san cua te bao ung th

Th nghiem phan lap va nuoi cay te bao ung th co t cung

1.2.2.2. Quan niem n dong

1.2.2.3. Thi gian nhan oi u

Th nghiem phan lap va nuoi cay te bao ung th co t cung

1.2.3. Nguyen nhan gay ra ung th

Th nghiem phan lap va nuoi cay te bao ung th co t cung

1.3. C S PHAN T CUA UNG TH

1.3.1. Cac gen gay ung th (oncogen)

Th nghiem phan lap va nuoi cay te bao ung th co t cung

Th nghiem phan lap va nuoi cay te bao ung th co t cung

1.3.2. Cac gen c che ung th (antioncogen)

Th nghiem phan lap va nuoi cay te bao ung th co t cung

1.3.2.2. Gen p53

1.3.2.3. Gen p73

Th nghiem phan lap va nuoi cay te bao ung th co t cung

1.3.3. Gen ieu hoa chet te bao theo chng trnh

Chu trnh te bao

S o 1: Hien tng chng trnh hoa s chet te bao

Th nghiem phan lap va nuoi cay te bao ung th co t cung

Kch thch tong hp p.21

Kch thch gen c che

tang trng te bao

Th nghiem phan lap va nuoi cay te bao ung th co t cung

Th nghiem phan lap va nuoi cay te bao ung th co t cung

Th nghiem phan lap va nuoi cay te bao ung th co t cung

1.4. MOT SO PHNG PHAP TACH VA NUOI CAY TE BAO UNG

1.4.2. Mot so phng phap tach te bao ung th t khoi u

Th nghiem phan lap va nuoi cay te bao ung th co t cung

Th nghiem phan lap va nuoi cay te bao ung th co t cung

1.4.2.3. Phng phap tach te bao bang enzym

Cat nho khoi mo thanh nhng manh nho khoang 2 3mm3.

Khuay t nhiet o 37oC. e at c mat o te bao can thiet th thi gian

Th nghiem phan lap va nuoi cay te bao ung th co t cung

1.4.3.1. Moi trng nuoi cay

Th nghiem phan lap va nuoi cay te bao ung th co t cung

1.4.3.2. Huyet thanh

Th nghiem phan lap va nuoi cay te bao ung th co t cung

1.4.3.3. Moi trng khong co huyet thanh

1.4.3.4. Moi trng vat chat

Th nghiem phan lap va nuoi cay te bao ung th co t cung

1.4.4. Nuoi cay s cap

1.4.5. Cay chuyen

Th nghiem phan lap va nuoi cay te bao ung th co t cung

1.4.6. Tao dong

1.4.7. Ky thuat ong lanh te bao

Th nghiem phan lap va nuoi cay te bao ung th co t cung

1.5. NHAN DANG DONG TE BAO UNG TH

Th nghiem phan lap va nuoi cay te bao ung th co t cung

1.5.2. ac iem phat trien cua dong te bao ung th

Th nghiem phan lap va nuoi cay te bao ung th co t cung

1.5.3. Xac nhan ac iem dong te bao ung th

Th nghiem phan lap va nuoi cay te bao ung th co t cung

1.5.4. Tap hp cac dong te bao ung th

1.6. Cac ng dung cua ky thuat nuoi cay te bao ung th

Th nghiem phan lap va nuoi cay te bao ung th co t cung