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 Tn hnh Trang

2.1 S tin trnh th nghim 17

1

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.

2

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.

3

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

4

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

5

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].

6

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.

7

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.

8

- 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: 150

o.

- 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

9

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 51Cr 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-Glu-

OH 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:

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.

3 4 5

CY (120 mg/kg)

i.p.

1 13 12

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

Carbon

(0,1 ml/10 g)

i.v.

nh

lng

Thi gian (ngy)

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 tuyn c tng i (%)

Ch s thc bo

Khi lng lch tng i (%) = Trng lng chut

Khi lng lch

100 %

Khi lng tuyn c tng i (%) = Trng lng chut

Khi lng tuyn c 100 %

K = T1 T2

Ln OD1 Ln OD2

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

L chut

Trng lng (g) S bin i

trng lng

(g) Ngy th 1

Ngy th 3

(s dng

CY)

Ngy th 13

Chng sinh hc 33,63 1,33 35 2,00 42,13 2,17** +7,13 2,17

Chng m 34,00 1,39 34,75 1,49 27,13 3,68 -7,62 3,02

Pidotimod liu

200mg/kg 33,75 2,60 34,88 2,59 30,63 2,50*

-4,25 0,46

Pidotimod liu

400 mg/kg 31,78 2,58 33,38 2,50 28,25 1,75

-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 N Trng lng lch tng

i

T l so vi

chng m

Chng sinh hc 8 0,523 0,118 0,68

Chng m 8 0,770 0,148 1,00

Pidotimod liu 200 mg/kg 8 0,654 0,112 0,85

Pidotimod liu 400 mg/kg 8 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 N Trng lng tuyn c

tng i

T l so vi nhm

chng m

Chng sinh hc 8 0,143 0,049 3,40

Chng m 8 0,042 0,029 1,00

Pidotimod liu 200 mg/kg 8 0,135 0,049** 3,21

Pidotimod liu 400 mg/kg 8 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 N S lng bch cu (103

t bo/ml) T l

Chng sinh hc 8 17,113 8,994 3,69

Chng m 8 4,638 1,036 1,00

Pidotimod liu 200 mg/kg 8 10,775 3,541** 2,32

Pidotimod liu 400 mg/kg 8 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 N Mt quang

ti 0 pht

Mt quang

ti 15 pht Ch s thc bo

T

l

Chng sinh

hc 8 0,082 0,048 0,041 0,026 0,053 0,026 3,12

Chng bnh

l 8 0,120 0,030 0,093 0,024 0,017 0,009

1,00

Pidotimod

200 mg/kg 8 0,123 0,039 0,069 0,015 0,037 0,013* 2,18

Pidotimod

400 mg/kg 8 0,111 0,020 0,067 0,018 0,036 0,018 2,12

*: 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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