FULL NAME: NGUYN TH HNG NHUNG

NI C T V NUI CY T BO CU 1. TM TT BI VIT: I. Gii thiu chung: S nhim ni c t ln cc sn phm thuc da trn t bo l mi quan tm hng u i vi ngnh cng nghip dc phm. Trong nui cy t bo, ni c t khng nhng nh hng n kh nng sinh trng m cn gy bin i chc nng ca t bo. Do vy rt cn thit phi nghin cu tm ra cc c tnh ca ni c t, cc ngun sn sinh v c bit l nh hng ca n ln nui cy t bo c bin php khc phc. II. Ni c t l g? L mt phc hp lipopolysaccarit (LPS)-thnh phn chnh ca lp mng ngoi t bo vi khun Gram m. Vi khun tit mt lng nh ni c t vo mi trng khi chng ang sinh trng mnh v tit mt lng ln khi chng cht. Cu to LPS: Bao gm 1 nhm lipit rt k nc lin kt cng ha tr vi ui polysaccarit di. Lipit A: gm 2 phn t ng phosphoryl ha (glucosamine) lin kt vi nhiu phn t axit bo. Lipit A gip gn cc phn t LPS vi mng t bo v chu trch nhim cho hu ht cc hiu ng sinh hc ca ni c t. ui polysaccarit di a nc gm 2 vng chnh: o Vng cha mt nhm nh ng: phn t ng 7 cacbon lin kt vi 2 phn t ng 8 cacbon, c vai tr lin kt lipit A vi khng nguyn O. o Vng ln hn v c bit d bin i O (khng nguyn O), gy ra cc phn ng khng nguyn c th trong c th ngi chng li vi khun Gram m. c tnh: Mang tnh k nc ln a nc Tch in m trong dung dch Bn nhit cao v c xu hng tp hp thnh phn t kch thc ln (1000KDa hoc hn ty thuc vo pH, nng mui v hot tnh b mt trong dung dch) C i lc ln vi cc b mt k nc, c bit l cc dng c bng nha dng trong phng th nghim. III. Phng php pht hin v nh lng Nhng nm 1940: pht hin ni c t bng cch quan st hin tng st khi tim vo th (t tin, tn thi gian v khng nh lng c) Nhng nm 1970: phn tch invitro, phng php LAL da vo s ng t ca dch in gii t bo bch cu cua mng nga Limulus polyphemus khi c mt mt lng ni c t rt nh, phng php ny nhy, pht hin c ni c t di 0.03 n v c t (EU)/mL (1 n v xp x 1-2ng ni c t/ml dung dch ty thuc vo cht chun c s dng). Ngy nay, phng php phn tch phn ng sinh mu hoc o c nhy hn LAL, pht hin ni c t nng di 0.001 EU/ml.

IV. Ngun sn sinh ni c t trong nui cy t bo 1. Nc: Do quy trnh x l nc cha vn hnh ng hoc nc tinh khit sau x l c tr trong cc bnh cha bng nha hoc thy tinh khng m bo lm cho nc b nhim ni c t. 2. Huyt thanh: Trc y huyt thanh thai nhi b (FBS) l mt ngun sn sinh c t, tuy nhin ngy nay vi phng php phn tch LAL gip cc nh sn xut loi tr ni c t khi FBS, hm lng ni c t cn li khong di 1 ng/mL. 3. Mi trng v cc cht ph gia: Ni c t trong mi trng do nhim t ngun nc v cc thnh phn khc (d c kh trng hoc lc cng khng th loi b ht c t, cc axit amin s dng c ngun gc t ln men vi khun cha ni c t). 4. Bnh cha bng nha v thy tinh: do cc phng php kh trng cha loi b c ni c t v ni c t c tnh bn nhit v bn vi chiu x, thm vo li c i lc cao vi b mt dng c bng nha v thy tinh. V. nh hng ca ni c t ln sinh trng v chc nng t bo Kch thch cc i thc bo v bch cu n nhn sn xut ra rt nhiu cytokine, bao gm TNF (yu t hoi t khi u) v cc interleukin trung ha c t, gy ra sc nhim khun v suy a c quan trong c th, kch thch h min dch sn sinh cc t bo T, hoc t bo B, cc lymphokine hoc interleukine-1,6,8, cc khng th trung ha cc ni c t. In vivo, ni c t lm nh hng n s co mch gy suy yu h tun hon. In vitro, cc t bo c ngun gc t mu rt mn cm vi ni c t cn t bo khng c ngun gc t mu th t mn cm hn. Lm thay i kh nng sinh trng, bit ha v cc sn phm protein ca t bo. Khng gy ra nh hng g ln 7 dng t bo (v d WI38, 3T3, CHO) Lm gim t l thnh cng ca th tinh nhn to d nng thp. Kich thch t bo tit ra cc yu t hot ha tiu cu (lipit) nh prostaglandin E2, Thromboxine A2 VI. C ch tc ng ca ni c t LBP l 1 glycoprotein kch thc 60 KDa lin kt vi LPS thng qua ui lipit A lm tng cng cc tng tc ca n vi t bo min dch. Th th CD14 l mt glycoprotein c bit kch thc 55 kDa nm trn b mt cc t bo min dch. Trong huyt thanh, khi LPS- LBP lin kt vi CD14 s khi ng cc tn hiu bn trong t bo, tc ng ln nhn lm tng cng phin m to mARN (nh hot ha MAP kinase) m ha cho cytokine, t rt nhiu phn t cytokine c tit ra ngoi t bo. Khi thiu cc th th CD14, LPS vn c th xm nhp vo t bo nh cc th th cha xc nh, bng thc bo hoc trc tip xm nhp qua mng vo t bo. Trong mt s trng hp chng c th lin kt vi b vi ng ca khung xng t bo, c ch qu trnh tng hp vi ng. Cu 2: Bi vit ny ng dng vic nui cy t bo nh th no? Bi bo ny cung cp rt nhiu thng tin v ni c t trong nui cy t bo: cc c tnh ca n, cc ngun sn sinh, c ch hot ng v c bit l nh hng ca n ln nui cy t bo gip cho cc nh nghin cu v nui cy t bo c th c bin php thch hp phng trnh ni c t.

Th nht, v phng php pht hin v nh lng, n cung cp cho ta 3 phng php gip pht hin nhanh, nhy v nh lng c hm lng ni c t ngay c khi n nng rt thp, l phng php LAL c th pht hin c ni c t vi hm lng 0.03 EU/mL, v phng php th phn ng sinh cht mu hoc o c nhy hn c LAL cho php pht hin ni c t ngay khi nng ca n nh hn 0.001 EU/mL. Nh c nhng phng php ny ta c th lm cc php th bt c khi no nghi ng c ni c t trong nui cy c bin php x l kp thi. Th hai, gip cho nh nghin cu nm r ni c t xut pht ch yu t nhng ngun no bit cch kim tra cng nh loi tr ni c t khu . V nc s dng cho nui cy: Cn nc tinh khit tuyt i, ch khu x l nc qua ct nha trao i ion do ni c t c i lc vi nha v thy tinh, nn cn test kim tra nu nghi ng, dng c cha nc cng cn c m bo. Trong trng hp khng th loi tr c t ra khi nc th c th s dng nc WTI (mt loi nc tinh khit dng tim) thay th. Huyt thanh: lu khi s dng huyt thanh, c bit l FBS, tuy nhin vi FBS c cc hng cung cp hin nay th c th tm thi yn tm v cht lng. Mi trng v cc cht ph gia: hu ht s dng mi trng c sn xut bi cc hng, khi nm c ngun gc ni c t xut hin trong mi trng, nh nghin cu nn ch ng sp xp cc test th nghim ni c t trc v c sau khi thm cc cht ph gia hoc c th yu cu nh sn xut xc nh mc c t trong sn phm ca h. Cc bnh cha bng thy tinh: do ni c t bm rt chc vo thnh cc bnh thy tinh, thm vo chng c tnh bn nhit rt tt nn rt kh loi b ni c t ra khi cc bnh cha khi x l nhit. Qua bi vit ta bit c quy trnh x l ni c t khi dng c bng thy tinh bng cch kh trng 2500C trong hn 30 pht hoc 1800C trong 3 gi. i vi cc bnh cha bng nha, kh s dng cch kh trng bng nhit cao nh thy tinh, trong khi ni c t li rt bn nn kh trng bng chiu tia ch tiu dit c vi sinh vt m khng ph hy c cu trc ni c t. Do vy, khi s dng cc ng ly tm, bnh cha, bnh nn, mng lc, pipet ly huyt thanh, a petri u phi m bo rng hm lng ni c t di 0.1 EU/mL.

Hy m t tng nghin cu trong tng lai ca bn lin quan n bi vit ny v vic nui cy t bo gc Mc d cha c tip xc nhiu vi k thut nui cy t bo gc, nhng bi vit ny gip em hnh dung ra nhng cng vic cng nh cch thc nghin cu phng trnh tc hi ca ni c t trong nui cy t bo. Qua bi bo ny, em bit c nh hng ca ni c t n nui cy t bo, c ch ca nh hng v cc phng php loi tr n. Trc tin l s kim sot nghim ngt nng ni c t c th xut hin trong cc dng c cng nh ha cht s dng trong nui cy bi ni c t rt d sinh ra v kh ph hy n bi cu trc rt bn v c i lc ln vi cc dng c bng nha hoc thy tinh do c tnh k nc. Nh vy, khi nui cy t bo gc, iu u tin l phi chun b cc nguyn liu v dng c tht tt, m bo hm lng ni c t mc cho php khng gy nh hng n s sinh trng v bit ha t bo, phn tch LAL kim tra ni c t tn ti trong mt s ngun pht sinh chnh nh nc, huyt thanh, mi trng, cht ph gia cng nh cc bnh cha bng nha v thy tinh, s dng cc phng php loi b ni c t thch hp i vi tng thnh phn c th nh nu trn. ng thi khi bit c c ch tc ng ca ni c t, ta s tm cch loi b tc hi ca n, nh vo 2 c ch chnh l s lin kt LPS vi protein v lin kt ca phc LPS-LBP vi th th, chng hn nh ph hy lin kt LPS vi protein bng ha cht hoc cc loi sc k (v d Triton X114, cc loi ankan, s dng sc k i lc polymixin B, histidin, sc k trao i ion..), chng khng c

tnh khng nguyn m ch tn ti nh mt hapten, trnh cc p ng min dch ca t bo, hoc nghin cu tm ra nhng cht khng c hi cho t bo m c th cnh tranh th th CD14 hoc cc th th xuyn mng vi ni c t. Cu 3. a ra nhng thng tin khng c nu trong bi vit ny nhng n gip cho ngi c hiu y bi vit ny V kh nng gy p ng min dch ca ni c t, Morrison v cc cng s gii thch nh sau: Khi LPS c gii phng ra khi b mt t bo Gram m, chng lin kt vi cc protein trong huyt thanh v huyt tng to phc LPS-LBP, phc ny c th lin kt vi th th CD14 dng ha tan (sCD14) hoc dng nm trn mng t bo (mCD14), hot kha cc tn hiu bn trong t bo. sCD14 kim sot min dch t bo biu m v ni m vi LPS cn mCD14 th kim sot min dch trong cc trng hp nhim trng huyt. Rietschel v cng s ch ra rng nh hng ca ni c t l do s tng tc gia ui lipid A v cc t bo bch cu n nhn, cc i thc bo thng qua to ra cc lipid (Prostaglandin E2,Thromboxane A2) v protein (TNF, IL-6) Mi trng nui cy t bo gc gm cc thnh phn sau: mui v c, carbohydrate, acid bo, amino acid, vitamine, yu t vi lng, huyt thanh. Mi thnh phn c chc nng khc nhau. o Mui v c: gi cn bng p sut thm thu ca cc t bo, iu ha in th mng. o Carbohydrate, acid bo, amino acid: cung cp cc cht dinh dng thit yu gip t bo phn chia. Trong , carbohydrate l ngun cung cp nng lng chnh cho t bo, thng l glucid. o Vitamine: c th lin quan n trng thi bit ha ca t bo trong s iu ha chc nng, hay hot ng nh mt cht chng oxy ha. Vitamin nhm B cn cho s tng sinh v pht trin ca t bo. Thng thng vitamine s dng trong mi trng l riboflavin, thiamine v biotin. o Yu t vi lng: bao gm km, ng, selenium... trong selenium l cht gip tch cc gc oxy t do. o Huyt thanh: cung cp cht dinh dng v cc nhn t tng trng, kch thch s phc hi cc tn thng ca t bo, chng oxy ha v lm tng tnh bm dnh ca t bo ln b mt bnh nui. Chnh nhng thng tin ny gip hiu r hn ti sao li nu ra nc, huyt thanh, mi trng v cc cht ph giali l nhng ngun chnh cha ni c t trong nui cy t bo. Theo cng trnh nghin cu ca tin s Michael E. Dawson ng trn LAL Update, ni c t lm cho cc t bo tit interferon, TNF gy nh hng n qu trnh tng hp cc protein ca t bo (v d interferon c ch tng hp estelase, TNF c ch tng hp lipase khi nui cy cc t bo tim), hay cc ni c t gy nh hng n qu trnh nguyn phn v c ch t bo phn chia, do vy nh hng n s sinh trng ca t bo.