B Y T

TRNG I HC DC H NI

L TH IM

TH NGHIM TC DNG KCH THCH

MIN DCH CA PIDOTIMOD SN
XUT TI VIT NAM TRN
NG VT TH NGHIM

KHA LUN TT NGHIP DC S I HC

H NI - 2014

B Y T
TRNG I HC DC H NI

L TH IM

TH NGHIM TC DNG KCH THCH

MIN DCH CA PIDOTIMOD SN
XUT TI VIT NA M TRN
NG VT TH NGHIM
KHA LUN TT NGHIP DC S I HC

Ngi hng dn:

TS. Nguyn Vn R
Ni thc hin:
1.
B mn Ha Sinh.
2.
B mn Dc Lc.

H NI 2014

LI CM N
Li u tin, ti xin by t lng knh trng v bit n su sc ti ngi thy: TS.
Nguyn Vn R trng B mn Ha Sinh trng i hc Dc H Ni tn
tnh hng dn, ch bo v ng vin ti trong sut qu trnh thc hin v hon thnh
kha lun tt nghip ny.
Ti xin chn thnh cm n ch ng Th Kim Khuyn hc vin cao hc kha
17 to iu kin, ch bo, gip ti trong sut qu trnh thc hin nghin cu.
Ti cng xin by t lng bit n chn thnh ti cc thy c, cc k thut vin
B mn Ha Sinh v b mn Dc Lc - trng i hc Dc H Ni to iu
kin gip ti trong sut qu trnh lm kha lun.
Ti cng xin gi li cm n ti cc thy c trng i hc Dc H Ni
nhng ngi thy du dt ti sut 5 nm hc va qua.
Cui cng, ti mun gi li cm n ti b m, gia nh, ngi thn v bn b,
nhng ngi lun st cnh ng vin v gip ti vt qua nhng lc kh khn
nht trong hc tp v qu trnh hon thnh kha lun.
H ni, ngy 10 thng 05 nm 2014
Sinh vin

L Th im

MC LC

CC K HIU V CH VIT TT
DANH MC BNG
DANH MC HNH
T VN ........................................................................................................ 1
CHNG 1. TNG QUAN .................................................................................. 2
1.1. Tng quan v sinh l min dch ................................................................................ 2
1.1.1. Khi nim v min dch ..................................................................................... 2
1.1.2. Min dch khng c hiu (non-specific immunity).......................................... 2
1.1.3. Min dch c hiu (specific immunity) ............................................................ 2
1.1.4. Cc c quan v t bo tham gia qu trnh MD ............................................ 2
1.1.5. Tng quan v sinh l bnh suy gim min dch: ............................................... 4
1.2. Cht kch thch min dch v nguyn l nh gi tc dng ...................................... 6
1.2.1. Khi nim ........................................................................................................... 6
1.2.2. C ch tc dng ca cc cht KTMD ................................................................ 6
1.2.3. Nguyn l mt s th nghim nh gi tc dng ca cht KTMD ................... 6
1.3. Mt s nghin cu v Pidotimod .............................................................................. 7
1.3.1. Cu trc ha hc ca Pidotimod ........................................................................ 7
1.3.2. Dc ng hc ca Pidotimod .......................................................................... 8
1.3.3. Tc dng dc l ca pidotimod ....................................................................... 8
1.3.4. c tnh v tng tc thuc ............................................................................. 13
CHNG 2. I TNG V PHNG PHP NGHIN CU ................... 15
2.1. i tng nghin cu ............................................................................................ 15

2.2. Nguyn vt liu v thit b .................................................................................... 15

2.2.1. Thuc v ha cht ......................................................................................... 15
2.2.2. Thit b .......................................................................................................... 15
2.3. Ni dung nghin cu ............................................................................................. 15
2.4. Thit k nghin cu ............................................................................................... 16
2.4.1. Chun b ........................................................................................................ 16
2.4.2. Tin hnh th nghim .................................................................................... 17
2.4.3. Mt s ch tiu nh gi trong th nghim .................................................... 18
2.5. Phng php x l s liu ..................................................................................... 19
CHNG 3. KT QU ...................................................................................... 20
3.1. S bin i trng lng chut ............................................................................... 20
3.2. nh hng ca Pidotimod ln trng lng lch tng i .................................... 21
3.3. nh hng ca pidotimod ln trng lng tuyn c tng i ............................. 22
3.4. nh hng ca pidotimod ln s lng bch cu ................................................. 24
3.5. nh hng ca Pidotimod ln chc nng ca TB ............................................... 25
CHNG 4: BN LUN ................................................................................... 27
4.1. V thit k nghin cu ............................................................................................ 27
4.2. V kt qu nghin cu............................................................................................. 29
KT LUN V KIN NGH ............................................................................. 34
TI LIU THAM KHO

CC K HIU V CH VIT TT
Con - A

Concanavilin - A

CY

cyclophosphamid

DNCB

Dinitrochlorobenzen

TB

i thc bo

MD

p ng min dch

IFN

Interferon

IL

Interleukin

Kl/tt

Khi lng/th tch

KTMD

Kch thch min dch

LPS

Lipopolysaccharide

PHA

phytohaemagglutinin

RCT

Nghin cu lm sng ngu nhin, c i chng

p.o

ng ung

s.c

Tim di da

i.p

Tim phc mc

i.v

Tim tnh mch

i.m

Tim bp

DANH MC BNG:
Bng

Tn bng

Trang

3.1

S thay i khi lng chut

20

3.2

Khi lng lch tng i

23

3.3

Khi lng tuyn c tng i

25

3.4

S lng bch cu

27

3.5

Ch s thc bo

29

DANH MC HNH:
Hnh
2.1

Tn hnh
S tin trnh th nghim

Trang
17

T VN
Hin nay, cc bnh lin quan n virus, lin quan n phn t gy suy gim
kh nng min dch ca c th nh: HIV, ung th, nhim trng ngy cng gia
tng. Vy lm th no hn ch s pht trin ca bnh cng nh ko di s sng
ca ngi bnh l vn lun c x hi quan tm.
Cht kch thch min dch c a vo s dng nh mt bin php gip
con ngi chng vi bnh tt. Cc nh khoa hc tm ra mt s cht kch thch
min dch c ng dng cao nh: BCG, bronchovaxom, lentinan, levamisol...
Pidotimod c cu trc ging dipeptid l mt ha dc c kh nng kch thch min
dch c tm ra bi hng Poli Industria Chimica Italia vo u nhng nm 90
ca thp k XX. Nhiu nghin cu tin lm sng v lm sng khng nh pidotimod
c tc dng tt trn h min dch. Hin nay, thuc c a vo sn xut, s
dng nhiu nc trn th gii vi cc bit dc nh adimod, imunorix, onaka,
pigitil, polimod
Vit Nam, Trng i hc Dc H Ni v ang nghin cu, tng hp
pidotimod lm nguyn liu sn xut thuc. Tuy nhin, cha c nghin cu th
hot tnh ca thuc ny trn h min dch. V vy, chng ti thc hin ti Th
nghim tc dng kch thch min dch ca Pidotimod sn xut ti Vit Nam
trn ng vt th nghim nhm mc ch:
nh gi c tc dng ca Pidotimod sn xut ti Vit Nam ln trng lng
chut, trng lng tng i cc t chc lympho (lch, c) v s lng bch cu
ngoi vi v chc nng i thc bo h li ni m.

CHNG 1. TNG QUAN

1.1. Tng quan v sinh l min dch
1.1.1. Khi nim v min dch
Min dch (immunity) l kh nng ca c th nhn bit, p ng v loi b
cc yu t l gy hi.
Khi b yu t gy bnh (khng nguyn) xm nhp, trc tin c th vn hnh
ngay mt s t bo v phn t sn c kp thi ngn chn, x l, sau to ra cc
t bo v phn t c hiu tng ng vi tng loi khng nguyn khc nhau loi
tr chng.
Phn loi MD: gm 2 loi
- MD t nhin (min dch khng c hiu).
- MD thu c (min dch c hiu) [3], [5].
1.1.2. Min dch khng c hiu (non-specific immunity)
c hnh thnh trong qu trnh tin ha ca ca ng vt chng li s
xm nhp, gy nhim ca cc vi sinh vt gy bnh. Min dch khng c hiu
khng li tr nh, kh n nh v t b sai st.
Cc m, t bo, phn t tham gia vo MD khng c hiu gm: da, nim
mc, cc t bo thc bo, h thng b th, IFN, [5].
1.1.3. Min dch c hiu (specific immunity)
L trng thi min dch xut hin do khng th c hiu tng ng vi tng
khng nguyn c to ra sau khi c th tip xc vi khng nguyn (ngu nhin
hoc ch ng). Min dch c hiu c kh nng nhn dng c hu ht khng
nguyn v li tr nh min dch.
Min dch c hiu gm min dch th dch v min dch qua trung gian t
bo vi s m nhim ca cc t bo lympho B v lympho T [5].
1.1.4. Cc c quan v t bo tham gia qu trnh MD
1.1.4.1. C quan lympho trung ng
L ni sinh sn v bit ha t bo lympho n trng thnh, kh nng x
l khng nguyn.

Ty xng: L c quan trung ng ca mi t bo mu, nhy cm vi tia x

v cc thuc chng phn bo.
Tuyn c: m nhim chc nng hun luyn, phn chia, bit ha, cc t bo
lympho T l nh mt s yu t ha tan (thymulin, thymosin 1, ) [5].
1.1.4.2. C quan lymopho ngoi vi
L ni t bo lympho tr ng v bit ha di tc ng ca khng nguyn.
Hch lympho: c coi nh mt ci lc i vi cc phn t l ngoi lai v cc
mnh vn t m hoi t, l trung tm ca s tun hon cc t bo lympho, ni t
bo tip xc vi khng nguyn.
Lch: c nhim v lc v d tr mu cho c th, ngoi ra cn l ni tp trung
khng ca khng nguyn vo c th bng ng mu.
Cc m lympho khng c v bc: nm ri rc nim mc ng tiu ha, h
hp, tit niu, [5]
1.1.4.3. Cc t bo tham gia qu trnh MD
Lympho bo: chim khong 20 - 30 % tng s bch cu trong mu, c vai tr
ch cht trong min dch c hiu. Khi khng nguyn xm nhp vo c th, s gp
phi hng lot c ch bo v t nhin khng c hiu nhm tiu dit hoc loi tr
chng ra khi c th. Nu khng nguyn vt qua c hng ro ny, v c thi
gian, chng s gp phi cc cht bo v c hiu trong MD c hiu. MD
c hiu xut hin s lm MD t nhin c tng cng [5].
T bo dit t nhin NK (natural killer): L mt tiu qun th t bo ging
lympho, c kh nng dit mt s t bo ch: t bo u, t bo vt ch nhim virus.
Chc nng quan trng ca NK l kim sot min dch, ngn chn s di c ca t
bo u qua mu, bo v c th chng li s nhim virus. Cc t bo NK c th hn
ch hoc lm trm trng hn cc phn ng min dch [6], [26], [29].
T bo thc bo n nhn (monocyte): ng vai tr phng th do kh nng
thc bo cc loi k sinh trng v vi khun. Cc TB cn c vai tr quan trng
trong s khi ng qu trnh min dch c hiu, chng cn thit cho vic hot ha
cc t bo lympho T v lympho B trc s xm nhp ca cc khng nguyn ngoi

lai. Bn cnh , TB cn tit ra cc cht cytokin nh IL-1, IL-6, TNF- khi pht
qu trnh vim khng c hiu [6], [11], [18], [19], [27].
Cc t bo mu khc: Bch cu a nhn trung tnh, bch cu i kim v
dng bo (mastocyte), Bch cu i toan u c vai tr quan trng trong MD
ca c th vi khng nguyn.
1.1.5. Tng quan v sinh l bnh suy gim min dch:
Suy gim min dch l tnh trng ca c th sng trong h thng min dch
hot ng yu, khng p ng c vi yu cu ca cuc sng bnh thng, dn
n khng chng li c vi cc vi sinh vt gy bnh m hu qu l c th b
nhim trng nng i n t vong hay ni cch khc l sinh ra rt t khng th c
hiu trong khi c th khc li sinh min dch khi t trong cng iu kin.
Suy gim min dch c chia lm hai loi:
Suy gim min dch bm sinh
Suy gim min dch bm sinh hay tin pht l do nhng bt thng mang tnh
di truyn, to ra nhng khuyt tt trong h thng min dch, c th l:
+ Suy gim min dch ngay t t bo gc chung cho c hai dng lympho bo
B v T.
+ Suy gim min dch bm sinh dng T: c 2 trng hp: suy gim nng dng
T do s suy gim hot ng ca tuyn c lm cho dng lympho bo T khng trng
thnh v bit ho c, do khng c min dch qua trung gian t bo. Hin tng
ny gi l hi chng George. Trng hp th hai l ri lon qu trnh hot ho ca
lympho bo T trng thnh.
+ Suy gim min dch bm sinh dng B: c th l do tn thng tu xng m
khng c bit ho dng lympho bo B hoc c th c sai st trong qu trnh hot
ho ca lympho bo B trng thnh dn n ri lon s tng hp cc khng th
dch th.
+ Suy gim min dch bm sinh dng cc t bo thc bo v sn xut b th
gy gim t bo thc bo v thiu ht b th.
Suy gim min dch mc phi

Suy gim min dch mc phi l mt trng thi bnh l rt hay gp, l mt
hin tng th pht sau nhiu bnh. Nht l cc bnh gy suy dinh dng, nhim
c, nh hng ca mt s thuc gy c ch min dch v do kt qu ca cc bnh
truyn nhim nh ngi l nhim virus HIV - mt bnh nan y ca thi i
Suy gim min dch th pht do suy dinh dng
Ngi ta thy r rng khi c th b suy dinh dng s xut hin trng thi
suy gim min dch c khng c hiu ln c hiu m c ch bnh sinh ra l do
thiu nguyn liu trong sinh tng hp cc cht, c bit l thiu m.
Suy gim min dch th pht do nhim trng
Trong tt c cc trng hp nhim khun (virus, vi khun, nm hay k sinh
trng). Nu ko di s gy suy dinh dng dn n suy gim min dch. Nhim
virus dn n ri lon p ng min dch v lm suy gim min dch d dn n cc
bi nhim cc bnh khc.
Nhim khun mn tnh, c bit l nhim vi khun ni bo nh bnh hi, lao
th bao gi cng gy ra suy gim min dch t bo.
ngi, trong cn bnh th k AIDS do nhim bi loi Retrovirus HIV-I v
HIV-II, chng c i tnh c bit vi phn t CD4 v Receptor vi mt s chemokin
c trn cc t bo c thm quyn min dch m ch yu l cc lympho bo Th v i
thc bo. Virus lm ly gii cc t bo TCD4 hoc bt hot chng, s lng t bo
TCD4 suy gim trm trng ngi nhim HIV (bnh thng t l TCD4/TCD8 l 2/1,
khi nhim HIV th c th ch l 0,5/1).
T gim st Th dn n suy gim min dch trm trng.
Suy gim min dch th pht do mt s bnh khc.
Cc bnh c tnh nh ung th, bnh mu c tnh v cc bnh v thn nh suy
thn, thn nhim m... u dn n suy gim min dch.
Ngoi ra cc c th gi, do c nhng thay i trong hot ng min dch,
ngi ta thy c nhng suy gim min dch r rt, do ngi gi thng thy
tng kh nng nhim khun, hay b ung th, mc bnh t min (thp khp)..., chnh
l do s suy gim min dch [5].

Nh vy, MD l mt qu trnh phc tp xy ra khi c s tip xc ca

khng nguyn ngoi lai v h min dch. iu ha hot ng ca h min dch, gi
cn bng ni mi cn c s tham gia v phi hp ca nhiu c quan, t chc v t
bo s gip cho c th chng li cc khng khuyn gy bnh.
1.2. Cht kch thch min dch v nguyn l nh gi tc dng
1.2.1. Khi nim
Cc cht KTMD l nhng cht a vo c th nhm bin i kh nng ca cc
t bo p ng vi khng nguyn hoc lm thay i s tng tc gia cc t bo
hay gia cc cht trung gian cn thit cho s to nn mt MD [3], [6].
1.2.2. C ch tc dng ca cc cht KTMD
Ch yu theo 2 c ch:
-

Tc ng vo qu trnh bin i, chuyn ha ca cc khng nguyn nh

tng tch ly khng nguyn, gim o thi khng nguyn, do ko di s c mt

ca khng nguyn trong c th v gy ra kch thch lin tc ging nh tim khng
nguyn liu nh nhng nhiu ln v ko di.
-

Tng tc vi cc t bo c thm quyn min dch: lm tng hin tng

thc bo, tng hot ha TB, tng s tip xc v ha mng gia TB v t bo

lympho truyn thng tin khng nguyn t TB sang t bo lympho sn xut
khng th quyt nh tnh c hiu ca khng th i vi khng nguyn [3].
1.2.3. Nguyn l mt s th nghim nh gi tc dng ca cht KTMD
Cht KTMD tc ng vo nhiu khu trong qu trnh min dch. V vy, cc
k thut nh gi tc dng ca cc cht KTMD rt a dng v c th thc hin theo
cc khu chnh sau y:
Khu x l khng nguyn thng qua hot ng ca TB:
- K thut c ch di tn TB.
- K thut in di TB.
- K thut c ch dnh bch cu.
- K thut c ch tri ging bch cu.
- o hot tnh thc bo v nh gi kh nng tiu ha ni t bo ca TB.

C th nh gi chc nng ca TB trong giai on cm ng min dch bng

cch xc nh t l % cc TB c n khng nguyn v s lng khng nguyn b
vy bt mi TB (ch s thc bo) [3].
Khu hiu ng ca p ng min dch: lympho bo T
- Th nghim chuyn dng lympho bo: da vo c tnh ca lympho T, khi
c mt ca khng nguyn c hiu, cc lympho bo mn cm phn ng bng cch
chuyn sang dng blast (t bo ln, bo tng phnh to, ht nhn ni r).
- Th nghim to hoa hng t nhin E (Rosette E): da trn kh nng to hoa
hng vi hng cu cu ca lympho T.
- Th nghim to hoa hng kp: thng kt hp ng thi th nghim to
hoa hng E vi th nghim to hoa hng EA (hng cu g E c bao ph bi
khng th khng hng cu g A to phc hp EA) [3].
Khu p ng min dch ca lympho bo B v khng th Ig
- K thut hunh quang min dch.
- K thut to hoa hng EAC: dng pht hin lympho B mang th th
dnh cho b th, s dng hng cu cu E c bao ph bi hmolysin khng th
IgM (A) v b th (C) thnh phc hp EAC.
- K thut Jerne: pht hin lympho bo B tit khng th. Thc hin trn mi
trng thch hoc mi trng lng, da trn nguyn l to qung dung huyt khi
cho t bo lympho tip xc vi hng cu cu ng thi vi s c mt ca b th.
- nh gi tng i lng khng th bng cch xc nh hiu gi khng th
trong huyt thanh da trn phn ng ngng kt hng cu [3].
Nh vy, i vi mt cht KTMD, c kh nhiu phng php nh gi tc
dng trn h min dch. Ty vo iu kin thc t cng nh i tng, mc tiu cn
nh gi la chn phng php ti u mang li hiu qu cao.
1.3. Mt s nghin cu v Pidotimod
1.3.1. Cu trc ha hc ca Pidotimod
Danh php
- Tn INN: Pidotimod.

- Tn

khoa

hc:

acid

(R)-3-[(S)-(5-oxo-2-pyrrolidinyl)carbonyl]-

thiazolidine-4-carboxylic.
- M ATC: L03AX05 [16], [19], [29].
Cng thc ha hc
- Cng thc phn t: C9H12N2O4S.
- Khi lng phn t: 244,26 g/mol.
- Cng thc cu to: [16], [19].

Tnh cht l ha
- Bt tinh th khng mi, mu trng hoc trng ng.
- im chy 194oC198oC, s chy km theo phn hy nhanh chng.
- Nng sut quay cc []25D: 150o.
- tan ca Pidotimod trong nc l 37,8 (g/l) [16].
1.3.2. Dc ng hc ca Pidotimod
Trn ng vt (chut, ch): Pidotimod hp thu nhanh qua ng ung, tc
thi tr nhanh (T1/2 = 1 gi), th tch phn b ln, thuc phn b nhanh vo cc c
quan chnh, c bit l gan v thn. S bi tit qua nc tiu dng khng chuyn
i l 75,6% sau tim tnh mch v 31,1% sau ung. Sinh kh dng ca thuc l
100% qua ng tim bp v 27% qua ng ung [13].
Trn ngi: Thuc hp thu nhanh qua ng ung v sinh kh dng l 40
44%, T1/2 = 4 gi, khng ph thuc liu v ng dng; thuc khng b chuyn ha
v thi tr hu ht dng cha cha chuyn ha ra ngoi nc tiu; th tch phn
b ln (khong 30 lt) [22].
1.3.3. Tc dng dc l ca pidotimod
Nhiu nghin cu trong ng nghim, trn ng vt th nghim v trn ngi
trc y cho thy pidotimod c tc dng dc l trn h min dch rt r rng, tc

ng trn nhiu t bo, nhiu khu ca qu trnh min dch. Trong khun kh ca
bi kha lun, chng ti ch trnh by mt cch ngn gn mt s kt qu ca cc
nghin cu v cc tc dng ca Pidotimod a ra ci nhn tng quan nht v tc
dng ca cht iu bin min dch ny.
1.3.3.1. Cc nghin cu trn ng vt
Tc ng ln TB
Cc th nghim trn chut b c ch min dch chng t pidotimod c tc ng
tch cc ln chc nng ca TB (kh nng ha ng ng, kh nng sn xut gc
c, sn sinh cc cytokin gy vim) [12], [27], [28].
- Pidotimod lm tng tnh ha ng ng ca TB: Prednisolon lm gim
kh nng ha ng ng ca cc TB xung cn so vi nhm chng (khng b c
ch min dch, p < 0.001), nhng khi iu tr vi pidotimod, kh nng ha ng ng
ca TB c tng cng mt cch r rt v phc hi gn nh hon ton so vi
nhm chng (k c ng ung v tim phc mc) [12], [28].
- Pidotimod kch thch TB tng cng sn xut cc gc c: S dng
prednisolon gy suy gim min dch lm gim ng k qu trnh sn xut cc gc
c ca TB khi c vi cc tc nhn thch hp (anion superoxid, p < 0,01; NO,
p < 0,001). V khi iu tr cng vi pidotimod th s c ch ny gn nh b loi b,
a lng NO, superoxid tr v mc ban u [12], [28].
- Pidotimod kch thch TB sn sinh ra cc cytokine gy vim: Khi c
hot ha, cc TB cng c th to ra nhng cytokin gy vim nh TNF-, IL-1 v
IL-6. Nghin cu trn chut b c ch min dch bi prednisolon v c kch thch
bi cc yu t gy vim nh LPS, bo t nm Aspergilus fumigatus s sn sinh
TNF- thp hn ng k so vi nhm chng (khng b c ch, p < 0,05) nhng khi
c iu tr bng pidotimod th lng TNF- c hi phc v thm tr cn tng
so vi nhm chng (p < 0,001) [13], [28].
Tc ng ln t bo NK
Nghin cu trn t bo NK lch chut cc tun tui khc nhau (10 tun v
30 tun) c vi t bo YAC-1 c gn ng v phng x

51

Cr cho thy,

10

pidotimod liu cao (200mg/kg) tng cng hot ng ca t bo NK (khng phn

bit so vi chng), liu thp hn thay i khng ng k, thm ch cn gy c ch
(liu 50 mg/kg). Kt qu ny cho thy pidotimod c kh nng iu bin hot ng
ca NK [26].
Tc ng ln t bo lympho
- Trn min dch qua trung gian t bo: Nghin cu cc phn ng to hoa
hng vi hng cu cu, phn ng tng sinh p ng vi cc mitogen (Con A,
LPS), phn ng qu mn mun vi DNCB hay phn ng chng thi ghp chut b
gy suy gim min dch v c iu tr bng pidotimod u tng ln c ngha
thng k so vi nhm khng c iu tr. Cc kt qu u th hin pidotimod c
kh nng phc hi cc chc nng ca h min dch qua trung gian t bo tr v mc
bnh thng [12], [27].
- Trn min dch dch th: nh gi MD dch th ca pidotimod, ngi
ta s dng hai phn ng vi hai khng nguyn khc nhau (LPS i vi min dch
th khng ph thuc tuyn c; hng cu cu 3% - i vi min dch dch th ph
thuc tuyn c) trn chut c MD vi cc khng nguyn ny o hiu gi
khng th. Da vo cc kt qu ny, c th ni rng, pidotimod c tc dng lm
tng cng MD vi cc khng nguyn ny, a hiu gi khng th tr v mc
bnh thng [12], [27].
Tc dng chng li vi sinh vt
MD chng vi sinh vt l chc nng quan trng v cn thit bo v c
th chng li cc vi sinh vt gy bnh. Cc nghin cu trn chut cho thy,
pidotimod c tc dng bo v, lm tng t l sng ca con vt trc tnh trng
nhim vi khun hoc virus.
-

Tc dng chng vi khun: nghin cu kh nng chng li vi khun

ca pidotimod, c nhiu m hnh thc nghim khc nhau c thit lp. Trong
c 5 m hnh nhm: xc nh liu trnh iu tr vi pidotimod cho hiu qu thp
nht; nh gi hiu lc tc dng ca thuc trn nhiu chng vi khun; so snh hiu
lc ca pidotimod vi nhng thuc c chn lm chun; nh gi tc dng ca

11

thuc trn chut b gy c ch min dch; nghin cu hiu lc ca thuc khi kt hp

vi cc khng sinh -lactam [14].
Cc nghin cu u ch ra rng, d cho khng c tc dng khng khun, khng
nm v khng candida iu kin in vitro, nhng pidotimod thc s c kh nng
kch thch c ch min dch khng c hiu ca c th vi hiu qu cao, tc dng
ca pidotimod l r rt v c ngha thng k cao sau 5 ln s dng trc tnh trng
nhim khun. Pidotimod chng li s ly nhim ca nhiu chng vi khun vi mc
liu 0,01 100 mg/kg i.p. 5 ln. Pidotimod c tc dng tng ng hoc tt hn
so vi cc thuc chun so snh nh bestatin, N-acetylmuramyl-L-Ala-D-iso-GluOH v tuftsin; th hin r tc dng bo v trn chut c gy c ch min dch,
cng nh gip tng cng tc dng khng khun in vivo ca cc khng sinh
cefotaxim v ampicillin, theo hng h tr hoc hip ng (khng sinh dit vi
khun trc tip v pidotimod tc ng ln h min dch lm tng kh nng tiu dit
vi khun ca c th) [14], [27].
-

Tc dng chng virus: Cc th nghim tin hnh vi 3 loi virus v 2

mc liu (virus) gy nhim khc nhau: virus vim mng no (Mengovirus), virus
Herpes simplex (HSV), v virus cm A chng PR 8. Pidotimod c dng 15
ngy trc khi gy nhim v trong sut qu trnh chut b nhim virus cho ti khi
cht. Tc dng ca thuc th hin t l sng st khc nhau gia l ch nhim virus
(l chng) v l nhim virus nhng c iu tr bng pidotimod (l th). Thi gian
m mt na s chut b cht l chng v l th: vi Mengovirus tng ng l 3
v 5,5 ngy; vi HSV tng ng l 3 v 5 ngy; vi virus cm A PR 8 l 3 v 6,5
ngy. Thi gian m tt c chut cht l chng l 7 ngy cn l th l 8 ngy
[17].
Pidotimod khng phi l thuc chng virus c hiu. Cc d liu nghin cu
ton cu ch ra rng, tc dng ca pidotimod khng ph thuc vo loi virus v
khng lin quan cht ch n c ch nhn ln ca mi loi virus [17], [27].
Cc tc dng khc

12

Ngoi tc dng trn h min dch, Pidotimod c nh gi kh nng tc

dng ln cc c quan khc ng vt th nghim.
Trn h thn kinh
- Pidotimod khng c tc ng ln h thn kinh[24].
Trn h tim mch
- C bn pidotimod khng nh hng ln s hot ng bnh thng ca tim
v huyt p; khng th hin tc dng chng tng huyt p hay chng lon nhp trn
chut th nghim [24].
Trn h tit niu
Cc thng s v th tch nc tiu v nng ion niu khng c s sai khc
vi bnh thng chut c cho dng pidotimod liu 100 v 200 mg/kg i.p v 200
mg/kg p.o. Duy ch c liu 400 mg/kg p.o nng ion Na+ v K+ c tng hn so
vi tr s bnh thng [24].
Cc kho st khc
Pidotimod khng c tc dng chng vim, khng gy h glucose mu, khng
gy t ti ch, khng nh hng ti kt tp tiu cu hay s p ng ca rut c lp
th vi histamin, acetylcholin v BaCl2 [24].
1.3.3.2. Cc nghin cu trn ngi
Cc nghin cu trn ngi nhm mc ch: xc nhn cc kt qu thu c
ng vt v kim tra cc tc ng trn h min dch ca pidotimod vi i vi cc
qun th bnh nhn c tnh trng suy gim min dch khc nhau.
-

Nghin cu trn cc bnh nhn b ung th, v bnh nhn b khi u (mi v

di cn) cho thy b sung Pidotimod lm tng p ng vi cc mitogen (con A,

PHA) v s tng tit IL 2 ca cc t bo n nhn vng ngoi vi v ca cc t bo
lympho T nhm bnh nhn ny.
-

Nghin cu trn nhm bnh nhn ln tui (c trng l suy gim min

dch qua trung gian t bo): pidotimod kch thch TB phc mc tng tit cytokine
IFN- v gim tit IL-6 so vi trc khi s dng v so vi nhm chng s dng gi

13

dc. Pidotimod cng kch thch t bo lympho T tng tit IL-2 v tng p ng vi
PHA ng k so vi nhm gi dc [27].
-

Trn i tng l tr em b nhim trng ng h hp, pidotimod lm

gim ng k tt c cc thng s thi gian st, iu tr khng sinh, t l ti pht

[26]. Nghin cu RCT trn 120 bnh nhn nhi b nhim trng h hp ti pht cho
thy, pidotimod lm gim 35% nguy c ti pht, gim thi gian nhp vin (86%)
v gim iu tr khng sinh (47%); p < 0,01. Nu ti pht c xy ra nhng bnh
nhn ny, cc triu chng ca bnh nhn cng gim nhanh hn [9].
-

Nghin cu RCT trn 60 bnh nhn nhi b nhim trng tit niu ti pht

cho thy, so vi gi dc, nhng bnh nhn s dng pidotimod c thi gian phc
hi nhanh hn (9,6 so vi 12,3 ngy); thi gian iu tr khng sinh ngn hn (6,9 so
vi 8,3 ngy); v mt s triu chng chnh (st, b tiu, tiu nhiu ln, tiu ra
mu) gim nhanh hn [11].
-

Trn bnh nhn b vim amidal cp tnh: pidotimod lm gim ng k tn

s cc bnh nhim trng cng nh ci thin cc du hiu lm sng (ho, kh nut) v

thi gian iu tr khng sinh.
-

Trn nhm bnh nhn Down b nhim trng ng h hp: pidotimod lm

gim ng k tn s, mc nghim trng v thi gian truyn nhim so vi nhm

chng khng c iu tr [27].
1.3.4. c tnh v tng tc thuc
Tng tc thuc: Pidotimod khng lm thay i nng v tc dng ca cc
thuc dng cng. Pidotimod kh an ton khi iu tr cng vi cc thuc khc [24].
c tnh: Nghin cu c tnh ca Pidotimod trn chut nht, chut cng,
ch, cho thy pidotimod kh an ton, c tnh thp.
-

c tnh cp th hin liu rt cao: i vi c 2 loi chut: > 8000 mg/kg

(p.o v i.p.), > 4000 mg/kg (i.v. v i.m). ch, liu c > 4000 mg/kg (p.o.), >
2000 mg/kg (i.v v i.m).
-

c tnh mn: theo di chut trong vng 4 thng (vi i.v.) v 12 thng

(vi p.o.), pidotimod khng th hin c tnh liu 200 mg/kg (i.v.) v liu 800

14

mg/kg (p.o.), liu ny cao gp 32,5 ln liu s dng trn lm sng. Tng t, theo
di c tnh trn ch 26 tun vi tim bp v 52 tun vi ng ung, pidotimod
khng gy c liu 300 mg/kg (i.m.) v liu 600 mg/kg (p.o.), cc liu ny cao
hn liu s dng trn lm sng ln lt l 48,8 v 24,4 ln.
-

Pidotimod khng nh hng ln chc nng sinh sn c chut ci v

chut c liu 600 mg/kg (p.o.) v 500 mg/kg (i.v.).

-

Pidotimod khng gy qui thai chut (1000 mg/kg i.v. v 600 mg/kg

p.o.), th (300 mg/kg p.o. v 500 mg/kg i.v.).

-

Ch s iu tr chut dao ng trong khong 40 80 [15].

Nh vy, cc kho st cho thy pidotimod kh an ton, ngoi tc dng trn h

thng min dch th s tc ng ti cc hot ng chc nng khc l khng c hoc
khng r rng. Tnh n nht trong tc ng ca Pidotimod (ch trn h min dch)
l mt thuc tnh c li trong iu tr, do hn ch c nhng tc dng ph
khng mong mun.

15

CHNG 2. I TNG V PHNG PHP NGHIN CU

2.1. i tng nghin cu
Chut nht trng, chng Swiss, khe mnh do Vin V sinh Dch t Trung
ng cung cp, khi lng trung bnh 20 2 g.
Tt c ng vt trn sau khi mua v c nui trong iu kin y thc n,
nc ung, m, v nh sng ti khu nghin cu ng vt thc nghim ca B
mn Dc Lc, trng i hc Dc H Ni. Chut c nui mt tun trc khi
tin hnh th nghim lm quen vi mi trng. Khi tin hnh th nghim chut
t khi lng 30 35 g.
2.2.

Nguyn vt liu v thit b

2.2.1.

Thuc v ha cht

Pidotimod do b mn Ha Dc, trng i hc Dc cung cp.

Cyclophosphamid (bit dc endoxan).

Mc pelikan 4001 (c).

HCl 0.1N.

Nc mui sinh l 0,9%.

2.2.2.

Thit b

Bm, kim tim, mao qun, panh, ko, bn m.

Cn k thut, cn phn tch.

My lc Vortex.

ng ng mu cha cht chng ng EDTA do bnh vin Mt H Ni

cung cp.

My m bch cu t ng (ti trng i hc Y H Ni).

Micropipett (0,5l 10 l).

My ly tm.

My o quang UV-VIS 2960, ti b mn Ha Sinh, trng i hc

Dc H Ni.

2.3.

Ni dung nghin cu

16

- Kho st nh hng ca pidotimod ln trng lng chut, trng lng

tng i ca cc c quan lympho (lch, c) v s lng bch cu mu ngoi vi.
- nh gi tc dng ca pidotimod trn chc nng ca TB h li ni m
bng phng php o thanh thi carbon (carbon clearance test).
2.4.

Thit k nghin cu
2.4.1.

Chun b

ng vt th nghim: Chut sau khi nui cho quen vi iu kin mi

trng mi, c tin hnh chia l ngu nhin, v nh. Chia chut th nghim
thnh cc l nh sau:
-

L 1 (n = 8) chng sinh hc: chut bnh thng, khng gy tn thng min

dch, khng c iu tr bng pidotimod.

-

L 2 (n = 8) chng m: chut b gy tn thng min dch bng

cyclophosphamid liu 120 mg/kg/ngy trong 3 ngy, tim phc mc v khng c

iu tr bng pidotimod.
-

L 3 (n = 8) pidotimod liu 200 mg/kg: chut b suy gim min dch bng

cyclophosphamid liu 120 mg/kg/ngy tim phc mc trong 3 ngy v c iu tr

bng pidotimod liu 200 mg/kg.
-

L 4 (n = 8) pidotimod liu 400 mg/kg: chut b gy suy gim min dch

bng cyclophosphamid liu 120 mg/kg/ngy, tim phc mc trong 3 ngy v c

iu tr bng pidotimod liu 400 mg/kg.
Thuc th
- Pidotimod liu 200 mg/kg: cn v pha pidotimod trong nc mui sinh l
thnh dung dch c nng 2% (kl/tt), liu ung l 0,1 ml/10 g th trng.
- Pidotimod liu 400 mg/kg: cn, pha pidotimod trong nc mui sinh l
thnh dung dch c nng 2,67% (kl/tt), liu ung l 0,15 ml/10 g th trng.
- S dng my lc Vortex ha tan hon ton.

Thuc gy suy gim min dch cyclophosphamid: s dng dng thuc

bt pha tim endoxan. Pha thnh dung dch c nng 1,2 % (kl/tt) trong nc
mui sinh l, tim phc mc vi liu 0,1 ml/10 g th trng.

17

Tt c thuc th v thuc c ch min dch c pha theo ngy, sau khi s

dng thuc tha c bo qun trong ngn mt t lnh.

Dung dch mc pelikan 4001 tim tnh mch: pha long mc theo t

l 1 ml mc : 8 ml nc mui sinh l [20].

Dung dch HCl 0,1N ly gii mu: ly 8,5 ml acid hydrochloric m

c pha vi nc va 1000 ml [1].

2.4.2.

Tin hnh th nghim

M hnh th nghim tin hnh theo th nghim ca Biozzi et al [8] c sa i

cho ph hp vi iu kin Vit Nam:

Carbon
(0,1 ml/10 g)
i.v.

CY (120 mg/kg)
i.p.

Thi gian (ngy)

12

Pidotimod (200 400 mg/kg, p.o.), 1 ln/ngy 12 ngy

13

nh
lng

Hnh 2.1. S tin trnh th nghim

Cch tin hnh:
- Theo di cn nng ca chut trong sut qu trnh cho ung thuc, cho
chut ung ng liu v theo di phn ng bn ngoi ca chut.
- Ngy th nht ca th nghim, cc l 3 v 4, chut c ung pidotimod
(liu 200 mg/kg v 400 mg/kg), 1 ln/ngy v ung trong 12 ngy lin tc.
- Cc ngy th 3, th 4 v ngy th 5 tnh t khi bt u cho ung Pidotimod,
cc l 2, 3, 4 c tim phc mc cyclophosphamid (liu 120mg/kg/ngy) gy
suy gim min dch.
- Vo ngy th 13 tt c cc c th chut c tim tnh mch keo mc
pelikan 4001 (carbon) pha long vi liu 0,1 ml/10 g th trng.

18

- Ti cc thi im 0 v 15 pht sau khi tim mc, ly mu mu t hc mt

mi chut cho vo tng ng chng ng ng ring bit, c nh du tng ng;
mu thu c em i m bch cu v o quang.
- Xc nh thanh thi carbon: mi mu c thm HCl 0.1 N ly gii
vi t l 10l mu/3ml HCl. Sau em ly tm vi tc 4000 vng trong 5 pht,
loi b cn v ly phn nc o quang, xc nh nng carbon trong mu. Tin
hnh qut ph xc nh bc sng ti c hp th cc i. y, thu c
bc sng cc i l 657 nm. Sau , o mt quang ti bc sng (mu trng
l mu chut khng tim mc, v tin hnh ly gii, ly tm nh cc mu khc).
- m bch cu: bng my m bch cu t ng.
- Xc nh trng lng lch v tuyn c: chut sau khi ly mu, git cht,
m bng bc l lch, tuyn c. Bc nh nhng ct ly ton b tuyn c, lch ca
tng con chut, dng gc thm kh nc v mu trc khi cho cc t chc ny ln
a cn cn.
2.4.3.

Mt s ch tiu nh gi trong th nghim

S bin i trng lng chut (SBTL)

SBTL = trng lng sau iu tr trng lng trc iu tr.
Khi lng lch tng i (%)

Khi lng lch

Khi lng lch tng i (%) =

Trng lng chut

100 %

Khi lng tuyn c tng i (%)

Khi lng tuyn c

Khi lng tuyn c tng i (%) =

Trng lng chut

Ch s thc bo

Ln OD1 Ln OD2
K=

T1 T2

100 %

19

OD1 v OD2 l mt quang o ti thi im T1 (0 pht) v T2 (15 pht) tng

ng.
2.5. Phng php x l s liu
Cc s liu nghin cu c x l thng k theo phng php t test Student.
S liu c din t di dng Xtb SD. S khc bit c ngha thng k khi p <
0,05.

20

CHNG 3. KT QU
3.1.

S bin i trng lng chut

Trong sut qu trnh tin hnh th nghim, trng lng chut c cn bng cn
k thut v ghi li tin theo di nh hng ca thuc n trng lng v cho
ung thuc ng liu. Trng lng chut, phn no ni ln nhng tc ng ca
thuc i vi c th.
SBTL = trng lng sau iu tr trng lng trc iu tr.
Kt qu s bin i trng lng chut trc v sau khi tim cyclophosphamid:
Bng 3.1. S bin i trng lng chut
Trng lng (g)

S bin i
trng lng

Ngy th 3

L chut
Ngy th 1

(s dng

Ngy th 13

(g)

CY)
Chng sinh hc

33,63 1,33

35 2,00

Chng m

34,00 1,39

34,75 1,49

33,75 2,60

34,88 2,59

31,78 2,58

33,38 2,50

Pidotimod liu
200mg/kg
Pidotimod liu
400 mg/kg

42,13 2,17**

+7,13 2,17

27,13 3,68

-7,62 3,02

30,63 2,50*
28,25 1,75

-4,25 0,46

-5,11 1,09

Du () th hin s gim khi lng, du (+) th hin s tng khi lng.

*: p 0,05; **: p 0,01 so vi nhm chng m.

: p 0, 05; : p 0,01 so vi trng lng trc khi gy bnh (ngy th 3).

Nhn xt:
- Trc khi s dng CY gy suy gim min dch (2 ngy u), trng lng
trung bnh ca cc l chut tng khng ng k, v gia cc l chut khc nhau
khng ng k (p > 0.05).

21

- Sau khi gy suy gim min dch v tip tc iu tr bng pidotimod (thi
gian l 10 ngy k t khi bt u s dng CY), trng lng chut c s thay i r
rt, trng lng chut l chng m gim so vi l chng sinh hc c ngha
thng k (p < 0,01). Trng lng chut l chng sinh hc tng ln 7,13 (g) so vi
thi im 3 ngy sau khi bt u th nghim, cn trng lng chut cc nhm c
s dng CY u gim ln lt l 7,62 g; 4,25 g; 5,11 g tng ng vi cc l chng
m, th liu thp v th liu cao.
- Sau thi gian iu tr, trng lng chut l pidotimod liu 200 mg/kg tng
12,9% c ngha thng k so vi nhm chng m (p < 0,05); trng lng chut
l th liu cao (400 mg/kg) tng 4,2% so vi l chng m (p > 0,05). Trng lng
tuy c phc hi nhng vn thp hn so vi mc ban u. Trng lng trung bnh
ca tng l chut trc v sau khi gy suy gim min dch khc nhau c ngha
thng k (p < 0,01).
- Sau iu tr, trng lng trung bnh gia 2 l th vi liu 200 mg/kg v 400
mg/kg khc nhau ng k (p < 0,05).
- Trng lng gia cc c th trong tng nhm chut c s chnh lch kh
nhiu, nht l sau thi gian iu tr (SD kh cao).
3.2. nh hng ca Pidotimod ln trng lng lch tng i
Lch l c quan lympho ngoi vi, ni tr ng ca cc t bo lympho. Vng ty
trng ca lch c cu to ch yu l cc m lympho b nh hng nhiu ca cc
thuc chng phn bo, gy suy gim min dch. nh gi trng lng lch tng
i (so vi trng lng chut gim bt sai s do s ph thuc gia trng lng
lch v trng lng c th) nhm xc inh nh hng ca pidotimod trn chut b
suy gim min dch. Lch sau khi c bc l v tch ring, c cn bng cn k
thut c sai s l 0,01 g.
Kt qu s khc nhau trng lng lch tng i gia cc nhm chut c
th hin trong bng sau:

22

Bng 3.2. Trng lng lch tng i

L chut

Trng lng lch tng

T l so vi

chng m

Chng sinh hc

0,523 0,118

0,68

Chng m

0,770 0,148

1,00

Pidotimod liu 200 mg/kg

0,654 0,112

0,85

Pidotimod liu 400 mg/kg

0,557 0,036**

0,72

*: p 0,05; **: p 0,01 so vi l chng m.

: p 0,05; : p 0,01 so vi l chng sinh hc.

Nhn xt:
- Cyclophosphamid lm tng ng k trng lng lch tng i chut nht
trng. Trng lng lch tng i ca l chng m tng 32% so vi l chng sinh
hc (p < 0,01).
- Pidotimod c hai liu c tc dng lm hn ch s gia tng ny:
Pidotimod liu 400 mg/kg c tc dng r rt hn, trng lng lch tng
i gim 28% so vi l chng m (p < 0,01), trng lng lch nhm ny cao hn
6,5% so vi nhm chng sinh hc, s khc nhau ny khng c ngha thng k (p
> 0,05).
L chut dng pidotimod liu 200 mg/kg, lm gim 15% trng lng
lch tng i so vi l chng m, tuy nhin s khc nhau gia 2 nhm ny khng
c ngha thng k (p > 0,05).
- S chnh lch gia cc c th trong cc l sinh hc, chng m, th liu thp
cng c s bin i ln (SD cao).
3.3. nh hng ca pidotimod ln trng lng tuyn c tng i
Tuyn c l mt tuyn quan trng trong h min dch, l mt c quan lympho
trung ng, ni hun luyn, phn chia, bit ha, cc t bo lympho T. Vng v
chim phn ln khi lng tuyn c, gm ch yu l cc t bo dng lympho, mt

23

s t bo biu m xen k v mt t TB. Do , chu nh hng nhiu ca thuc

chng phn bo nh CY. nh gi s khc nhau v trng lng tuyn c tng i
gia cc l chut xem xt mt phn nh hng ca cc thuc ln cu to v chc
nng ca tuyn c.
S khc nhau v trng lng tuyn c tng i gia cc l chut c biu
din trong bng sau:
Bng 3.3. Trng lng tuyn c tng i

L chut

Trng lng tuyn c

T l so vi nhm

tng i

chng m

Chng sinh hc

0,143 0,049

3,40

Chng m

0,042 0,029

1,00

Pidotimod liu 200 mg/kg

0,135 0,049**

3,21

Pidotimod liu 400 mg/kg

0,134 0,025**

3,19

*: p 0,05; **: p 0,01 so vi l chng m.

: p 0,05; : p 0,01 so vi l chng sinh hc.

Nhn xt:
-

Trng lng tuyn c tng i l chng m gim r rt so vi l chng

sinh hc, gim 3,4 ln so vi l chng sinh hc ( p < 0,01).

-

Cc l th dng pidotimod c 2 liu 200 mg/kg v 400 mg/kg u lm tng

trng lng tuyn c tng i so vi l chng m, ln lt tng ln 3,21 v 3,19

ln so vi l chng m (p < 0,01).
-

Trng lng tuyn c tng i hai l dng pidotimod vi liu 200 mg/kg

v l 400 mg/kg ln lt t 94,4% v 93,7% so vi l chng sinh hc. S khc

nhau v trng lng tuyn c tng i gia cc l chng sinh hc, hai l dng
pidotimod khng c ngha thng k (p > 0,05).

24

Khong dao ng ca trng lng tuyn c tng i gia cc c th trong

mt nhm kh ln (SD cao).

3.4. nh hng ca pidotimod ln s lng bch cu
Bch cu l thnh phn khng th thiu trong h min dch. l cc t bo
m nhim chc nng chnh trong vic bo v c th trc nguy c xm nhp ca
cc khng nguyn ngoi lai c hi. Mt h min dch khe mnh phi m bo s
lng v cht lng ca cc t bo ny. V vy s lng bch cu thng c s
dng nh gi v tc dng ca mt thuc trn h min dch. Mc d khng phi
l mt ch s c hiu nh gi tc dng ca mt thuc, nhng li l mt ch s
quan trng trong nh gi p ng chung ca h min dch. i vi pidotimod cng
vy, s lng bch cu trong mu ngoi vi l mt trong cc ch s dng nh gi
nh hng ca thuc trn h min dch.
S lng bch cu trong mu ngoi vi c m bng my m bch cu t
ng, kt qu c biu din trong bng sau:
Bng 3.4. S lng bch cu

L chut

S lng bch cu (103

t bo/ml)

T l

Chng sinh hc

17,113 8,994

3,69

Chng m

4,638 1,036

1,00

Pidotimod liu 200 mg/kg

10,775 3,541**

2,32

Pidotimod liu 400 mg/kg

12,912 5,780**

2,78

*: p 0,05; **: p 0,01 so vi l chng m.

: p 0,05; : p 0,01 so vi l chng sinh hc.

Nhn xt:
-

T bng trn ta thy, s dng CY lm gim r rt s lng bch cu trong

mu ngoi vi, gim 3,69 ln so vi l chng sinh hc (p < 0,01).

25

Cc l s dng thuc pidotimod iu tr, s lng bch cu c tng ln

ng k so vi l ch s dng CY. S lng bch cu l pidotimod liu 200

mg/kg tng 2,32 ln so vi l chng m (p < 0,01); l pidotimod liu 400 mg/kg
tng 2,78 ln so vi l chng m (p < 0,01).
-

S lng bch cu hai l pidotimod liu 200 mg/kg v 400 mg/kg ln lt

bng 62,96 % v 75,45% so vi chng sinh hc. S lng bch cu l pidotimod

liu 200 mg/kg thp hn ng k so vi chng sinh hc (p < 0,05).
-

Pidotimod liu 400 mg/kg c tc dng mnh hn so vi pidotimod liu 200

mg/kg, tuy nhin, s lng bch cu 2 l ny khc nhau khng c ngha thng
k (p > 0,05).
-

S chnh lch v s lng bch cu gia cc c th trong mt l kh ln (SD

cao) nht l l chng sinh hc, SD = 8,994. S chnh lch ln ny th gy ra

nhiu sai s khi so snh, thng k. C th do s lng chut trong mt l tng i
t (N=8) gy ra s chnh lch ny.
3.5. nh hng ca Pidotimod ln chc nng ca TB
TB l t bo bch cu n nhn, h thng cc TB trong c th cn c
gi l h thng li ni m gi vai tr quan trng trong c h min dch khng c
hiu v min dch c hiu. TB c kh nng ha ng ng v thc bo. Khi c th
b khng nguyn xm nhp s hot ha cc TB n thc bo v tiu ha cc
khng nguyn. ng thi, cc TB s truyn li cc thng tin v khng nguyn cho
cc t bo lympho to thnh tr nh min dch, gip nhn din v chng li cc
khng nguyn ny trong cc ln xm nhp tip theo. V vy, h thng li ni m l
hng ro c lc trong vic bo v c th trc nguy c bnh tt. nh gi kh nng
thc bo l mt trong cc test dng nh gi hot ng, chc nng ca h thng
li ni m.
Ch s thc bo (K) c tnh da vo nng carbon trong mu ti hai thi
im khc nhau (0 pht v 15 pht) trong php o thanh thi carbon.
Kt qu php o ch s thc bo nh sau:

26

Bng 3.5. Ch s thc bo

L chut

Mt quang
ti 0 pht

Mt quang
ti 15 pht

Ch s thc bo

T
l

Chng sinh
hc

0,082 0,048

0,041 0,026

0,053 0,026

3,12

Chng bnh
l

0,120 0,030

0,093 0,024

0,017 0,009

1,00

0,123 0,039

0,069 0,015

0,037 0,013*

2,18

0,111 0,020

0,067 0,018

0,036 0,018

2,12

Pidotimod
200 mg/kg
Pidotimod
400 mg/kg

*: p 0,05; **: p 0,01 so vi l chng m.

: p 0,05; : p 0,01 so vi l chng sinh hc.

Nhn xt:
-

T bng v hnh trn cho thy s dng CY lm gim ng k thanh thi

carbon trong mu. Ch s thc bo ca l chng m thp hn 3,12 ln so vi l

chng sinh hc (p < 0,01).
- L s dng Pidotimod liu 200 mg/kg c ch s thc bo tng ln ng k
so vi l chng m, tng ln 2,18 ln (p < 0,05); liu 400 mg/kg, ch s thc bo
tng ln 2,12 ln so vi l chng m (p > 0,05).
- Ch s thc bo l s dng pidotimod liu 200 mg/kg t 69,8%, l dng
liu 400mg/kg gim 67,9% so vi l chng sinh hc (p > 0,05).
- Ch s thc bo hai l dng pidotimod vi 2 mc liu (400 mg/kg v 200
mg/kg) khc nhau khng ng k (p > 0,05).
- S chnh lch v ch s thc bo gia cc c th trong nhm kh ln (SD
cao), nht l nhm chng sinh hc (SD = 0,026).

27

CHNG 4: BN LUN
4.1. V thit k nghin cu
C nhiu phng php nghin cu nh gi kh nng iu ha min dch ca
mt thuc. Da trn cc nguyn l khc nhau, ty thuc vo mc tiu nh gi m
s dng cc phng php nh nu trong mc 1.2.3. y, chng ti s dng
phng php o thanh thi carbon nh gi chc nng ca TB h li ni m
v s dng cc ch s v trng lng c th, trng lng lch tng i, trng
lng tuyn c tng i v s lng bch cu kho st tc dng KTMD ca
pidotimod. Phng php ny c s dng kh nhiu trn th gii nh gi tc
dng trn h min dch ca nhiu thuc. So vi cc phng php khc, phng
php ny thc hin kh n gin v r tin, kt qu c chnh xc cao.
Sau khi nghin cu mt s bo co th tc dng trn h min dch ca cc
thuc khc nhau v phng php ny [8], [12], [14], [20], [22], chng ti s
dng cyclophosphamid lm cht gy suy gim min dch, s dng mc pelikan
4001 lm khng nguyn ngoi lai, s dng phng php o quang ti bc sng
nh sng xc nh nng carbon v tin hnh nh gi trn cc ch s: khi
lng chut, khi lng lch tng i, khi lng tuyn c tng i, s lng
bch cu ngoi vi, ch s thc bo.
Phn ln cc cht kch thch min dch (immunostimulant) hoc iu bin
min dch (immunomodulatory) khng c hoc t c tc dng i vi h min dch
c cu trc v chc nng bnh thng, n ch c tc dng r rt i vi h min dch
b tn thng, suy gim hay ri lon [4]. Do vy, nghin cu tc dng kch
thch min dch ca Pidotimod, chng ti s dng m hnh nghin cu tc dng ca
thuc trn chut nht trng b gy suy gim min dch bng Cyclophosphamid.
C nhiu thuc c th gy suy gim min dch thng c dng trong
nghin cu nh cyclophosphamid, cycloprorin A, methotrexat, cc thuc chng
vim steroid (dexamethason, prednisolon) v yu cu chung t ra l cc thuc ny
phi gy suy gim min dch mt cch r rt so vi nhm i chng. Do , ty vo
i tng nghin cu v mc tiu nghin cu s dng thuc gy suy gim min

28

dch no cho hp l. i vi th nghim ny, chng ti s dng

cyclophosphamide gy suy gim min dch chut vi liu 120 mg/kg trong 3
ngy lin tip v kt qu l l chng m c s lng bch cu v cc ch s khc
gim r rt so vi l chng sinh hc.
CY l nm trong danh mc thuc thit yu, c xp vo nhm thuc c
ch min dch, c tc dng alkyl ha ADN, do c tc dng ngn chn s sao
chp v phin m ADN. CY c ch chung s phn chia ca tt c cc t bo ang
tng sinh, c bit l cc t bo ty xng v cc t bo biu m ng tiu ha. V
vy gy ra nhng tc dng khng mong mun trn nhiu c quan v m. CY gy
gim khng chn lc tt c cc dng t bo, k c bch cu, hng cu, tiu cu. S
lng bch cu trong mu thp nht thng thy sau 10 12 ngy iu tr [2]. CY
c ch s iu tr cao. LD50 ca thuc chut dao ng t 370 mg/kg (s.c) ti 360
mg/kg (i.p), nhanh chng b chuyn ha v bi tit trong vng 20 30 pht. Sau khi
tim phc mc c n 75% c bi tit trong vng 5 8 gi. iu tr CY chut
trong thi gian di s lm thay i cu trc ca mt s c quan trong c th nh
gan, lch, tuy,... [20]. Do vy, CY thch hp trong nghin cu nh hng ca thuc
ln cu to, chc nng ca cc c quan lympho v cc t bo bch cu.
Cc TB, ty vo hnh thi, tnh cht v ni tr ng m c tn gi khc
nhau: t bo Langerhans (da), Kuffer (gan), TB ph nang (phi) H thng cc t
bo ny cn c gi l h thng li ni m. nh gi chc nng ca h thng
li ni m, chng ti s dng ch s thc bo (phagocytic index) c tnh thng
qua thanh thi carbon (carbon clearance test) theo nghin cu ca Biozzi et al
[8]. Mt s tiu ch trong vic la chn cht dng nghin cu hot ng ca cc
t bo RES:
-

Cc ht phi c thc bo bi cc t bo RES v khng tng tc vi cc

t bo khc.

Khng i qua c hng ro mao mch.

ng nht v kch thc.

Khng c hi cho c th v cc t bo RES.

29

n nh in vitro v in vivo.

c o chnh xc [19].

V vy chng ti s dng mc pelikan 4001(c) thay cho vic s dng mc

Tu nh trong nghin cu ca Coppi G. et al [12]. So vi mc Tu th mc pelikan
c thnh phn v cht lng m bo chun hn, thnh phn chnh l carbon l mt
cht tr, khng c cc thnh phn khc gy p ng sinh hc. Cc phn t carbon
c kch thc mn (khong 250 ), ng u, to dng hn dch ng nht, khng
gy lng cn [8].
Phng php o quang l phng php kh d thc hin v tng i chnh
xc trong vic xc nh nng ca mt cht trong dung dch. i vi nghin cu
ny, chng ti tin hnh ly gii cc t bo bng HCl gii phng cc ht carbon
nm trong cc t bo khc ngoi h thng li ni m, sau ly tm loi b xc
t bo, thu c dung dch trong sut, khng nh hng n hp th ca carbon
v tin hnh qut ph (s dng mu trng l mu mu chut khng tim mc ti 2
thi im 0 v 15 pht) tm bc sng c hp th cc i ca carbon v thu
c max = 657 nm nh sng , ph hp vi cc nghin cu trc cng s
dng carbon clearance test tnh ch s thc bo.
tin hnh nh gi tc dng ca pidotimod, chng ti s dng cc ch s v
trng lng chut, trng lng tng i ca cc c quan lympho, s lng bch
cu v ch s thc bo. Cc ch s ny u lin quan t nhiu n kh nng min
dch ca c th. Trng lng c th, trng lng tng i ca cc c quan
lympho, s lng bch cu cho mt ci nhn tng quan v kh nng MD chung
ca con vt khi b gy suy gim min dch trc cc tc ng bn ngoi. Ch s thc
bo c trng cho chc nng v hot ng ca TB trong h thng li ni m.
4.2. V kt qu nghin cu
S dng CY lm gim ng k hu ht tt c cc ch s dng trong nghin cu
ny, gy ra s khc bit ng k i vi nhm i chng sinh hc. iu ny gip
nh gi r rng hn tc dng ca Pidotimod trn h thng min dch.

30

V trng lng chut, CY gy c ch min dch, c ch s tng sinh ca hu

ht cc t bo, c bit l t bo ty xng v t bo nim mc ng tiu ha, do
gy ra nhiu tc dng ph trn ng tiu ha v cc m, c quan khc. Qua
quan st, cho thy, cc l chut c s dng CY cc phn ng thng ngy chm
chp hn, thng nm tp chung mt ch, t hot ng v n t hn so vi l chng
sinh hc, mt s chut cn c biu hin tiu chy nhng ngy cui ca t dng
thuc. C l v vy m khi lng chut nhng nhm ny gim nhiu so vi ban
u, nht l l chng m, trong khi l chng sinh hc, khi lng chut vn tng
sau 13 ngy iu tr. Cc l s dng pidotimod c trng lng trung bnh tng so
vi l chng m, tuy nhin, mc tng ny cha tr v con s ban u trc khi gy
bnh v cng thp hn r rt so vi l chng sinh hc, c th do thi gian s dng
ngn nn cha t c mc ban u. Chng t, pidotimod c 2 mc liu c tc
ng ln trng lng chut.
V trng lng lch tng i, l chng m c gi tr ln nht, cn l chng
sinh hc c gi tr b nht, tc l CY lm tng trng lng lch tng i chut
nht trng. Lch c cu to bi ty trng v ty . Trong ty chim 4/5
khi lng lch, l m chn ca cc t bo hng cu gi hay tn thng v cc
mnh t bo cht. CY lm suy gim min dch nn con vt d b nhim cc tc nhn
gy bnh (vi khun, virus, k sinh trng...) v tng s lng cc t bo cht. y
cng c th l nguyn nhn lm tng khi lng lch nhng l dng CY. Ty
trng chnh l ni cha cc nang lympho nhng ch chim khi lng b (1/5 khi
lng lch) nn CY c lm gim trng lng lch th cng khng r rng. Do
nn tin hnh gii phu tuyn lch quan st s thay i ca phn ty trng, t
nh gi chnh xc hn MD ca c th b suy gim min dch. Tuy nhin, iu
khng c thc hin trong nghin cu ny, cng l mt trong nhng thiu
st cn c khc phc trong cc nghin cu tip theo. i vi ch s ny,
pidotimod cc liu cng lm hn ch mc tng trng lng a v gi tr so
snh. Mc d, liu 200 mg/kg mc gim ny khng c ngha thng k nhng

31

cng c th ni c rng, pidotimod c nh hng n trng lng tng i ca

lch.
V trng lng tuyn c tng i, nhm chng m c gi tr thp hn ng k
so vi nhm chng sinh hc, chng t CY lm teo nh c quan lympho ny. Tuyn
c l c quan lympho trung ng, l ni chn v bit ha ca lympho T. Tuyn c
teo nh nh hng n chc nng min dch, c bit l dng bch cu lympho T v
p ng to khng th ph thuc t bo T (T-dependent antibody response) ca
dng t bo lympho B. Cc l s dng pidotimod c khi lng tuyn c ln hn
ng k so vi l chng m v c s khc bit khng ng k so vi l chng sinh
hc. C th gii thch rng, pidotimod lm hn ch qu trnh apoptosis (cht theo
chng trnh) ca cc t bo tuyn c gy ra bi CY. Kt qu ny cng c bo
co trc y, khi t bo tuyn c vi cc tc nhn gy ra hin tng apoptosis
nh Ca++ - ionophores A23187, genistein, IL-2, v pidotimod; sau nh lng
s phn mnh ADN. Cc kt qu u th hin, pidotimod c kh nng bo v cc t
bo tuyn c khi hin tng phn mnh ADN gy ra bi cc tc nhn trn [25].
Nh vy, c th ni rng, Pidotimod c 2 mc liu c tc dng bo v tuyn c
di tc dng ca CY, gp phn lm tng MD nhng c th b suy gim min
dch.
V s lng bch cu, cng thy r s gim bch cu l chng m do s c
ch ty xng ca CY. cc l iu tr bng pidotimod lm hn ch ng k s
gim ny, d cha tr v mc bnh thng nh l chng sinh hc. C th gii thch
rng, pidotimod c tc dng ngn chn s alkyl ha ADN ca cc t bo ty xng
ca CY, bo v cc t bo ny khi hin tng cht theo chu trnh nhm duy tr
chc nng ca chng trong h min dch ni chung v h to mu ni chung. Cng
c th, pidotimod tc ng trc tip ln cc t bo bch cu hn ch s alkyl ha
ADN ca chnh cc t bo ny gy ra bi CY.
i vi ch s thc bo, y l thng s quan trng nh gi chc nng ca h
thng li ni m hay h thng cc t bo thc bo n nhn. Kt qu cho thy, tr
s ny gim mt cch ng k l s dng CY gy suy gim min dch (ch

32

bng 32% l chng sinh hc). C ngha l CY lm gim thanh thi ca carbon.
Khi tim tnh mch carbon vo trong mu, y l mt cht l i vi h min dch.
Khi h thng min dch s huy ng cc t bo min dch chng li tc nhn l
ny. H thng cc t bo n nhn cng vy, chng thc hin chc nng bt gi cc
ht carbon trong mu, do s lm nng carbon trong mu. Da vo kt qu trn
ta thy, nng carbon trong mu ca l chng m gim t hn (ch s thc bo
thp) so vi cc l cn li, chng t rng, hot ng ca cc t bo n nhn ca h
thng li ni m nhm ny c suy gim. cc l th dng pidotimod, ch s
thc bo tng ng k so vi l chng m ch ra rng thuc c tc dng trn h
thng li ni m, kch thch hot ng ca cc t bo thc bo n nhn, chng li
cc tc nhn l c hi cho c th. cc nghin cu trc trn tc dng ca
pidotimod, ch s thc bo ca l chng m, l chng sinh hc, l th liu 200
mg/kg v 400 mg/kg ln lt l 0,297 0,0079; 0,363 0,0266; 0,365 0,0114;
0,365 0,0119. Nh vy, ch s thc bo l chng m bng 98,07 % so vi
chng sinh hc (p < 0,05), v cc l th dng pidotimod lm tng ch s thc bo
ln 22,9 % so vi chng m, t 100,55 % so vi l chng sinh hc [12]. Trong th
nghim ny trn pidotimod sn xut ti Vit Nam, ch s thc bo l chng m ch
bng 32,08% so vi chng sinh hc (p < 0,05), cc l s dng pidotimod (liu
200 mg/kg v 400 mg/kg, p.o.) tng hn so vi chng m 117,65% v 111,16%, t
69,8% v 67,9% so vi l chng sinh hc. S khc bit ny c th do vic s dng
ha cht khc nhau, tin hnh trong cc mi trng v iu kin khc (th nghim
trc dng mc tu vi pha long l 0,32 ml mc : 1 ml nc mui v o quang
640 nm). C th ni rng, nu cng b suy gim min dch th pidotimod kch
thch lm tng min dch cng mnh.
V lch chun trong cc kt qu ca cc ch s nh gi: trong 5 ch s
c nghin cu, u cho lch chun kh cao. C th hiu l, s phn tn kt
qu nh lng gia cc cc th trong cng mt nhm kh ln. Ni cch khc, p
ng i vi thuc gia cc c th trong cng mt nhm l khc nhau. lch chun
cao c th do s lng mu trong th nghim tng i t (N = 8). lch chun

33

cao, c kh nng dn n sai s ln trong thng k, do sai s chun c tnh bng

cng thc sau [4]:

Nh vy, c th gim sai s cho php thng k bng cch tng s lng mu ln
nhiu ln. V mt s l do m nghin cu ny cha thc hin c vi c mu ln
hn, hy vng cc nghin cu tip theo s thc hin c iu ny kt qu c
ngha hn.

34

KT LUN V KIN NGH

Kt lun
Th nghim pidotimod hai mc liu 200 mg/kg v 400 mg/kg chut nht
trng, thu c nhng kt qu sau:
- Pidotimod c hai mc liu u lm tng trng lng chut so vi l
chng m: l dng liu 200 mg/kg tng 12,9% (p < 0,05), l dng liu 400 mg/kg
tng 4,2% (p > 0,05). L dng pidotimod liu 200 mg/kg lm tng trng lng
chut so vi l chng m c ngha thng k.
- L chut dng pidotimod liu 400 mg/kg lm gim trng lng lch tng
i 28% so vi l chng m c ngha thng k (p < 0,01), l dng liu 200 mg/kg,
lm gim 15% trng lng lch tng i so vi l chng m khng c ngha
thng k (p > 0,05).
- Trng lng tuyn c tng i ca l pidotimod liu 200 mg/kg v 400
mg/kg ln lt tng ln 3,21 v 3,19 ln so vi l chng m (p < 0,01) v t 94,4%
v 93,7% so vi l chng sinh hc.
- S lng bch cu l th dng pidotimod liu 200 mg/kg v 400 mg/kg
ln lt tng 2,32 v 2,78 ln c ngha thng k so vi l chng m (p < 0,01).
- Pidotimod lm tng ch s thc bo so vi l chng m: vi mc liu 200
mg/kg, tng 2,18 ln (p < 0,01); liu 400 mg/kg tng 2,12 ln (p < 0,05). c hai
mc liu s tng ch s thc bo c ngha thng k.
xut v kin ngh:
Cn lm thm cc nghin cu c quy m vi s lng mu ln, p ng yu
cu nh gi ton din v chnh xc tc dng kch thch min dch ca pidotimod
sn xut ti Vit Nam, sm c th a vo bo ch v sn xut thuc ny phc
v cho cng tc y t.

TI LIU THAM KHO

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