Tổng quan một số mô hình nghiên cứu tác dụng cầm máu của thuốc trên động vật thực nghiệm.pdf

B Y T

TRNG I HC DC H NI

PHM TH THU

TNG QUAN MT S M HNH

NGHIN CU TC DNG CM MU

CA THUC TRN NG VT THC

NGHIM

KHA LUN TT NGHIP DC S

H NI 2014

B Y T

TRNG I HC DC H NI

PHM TH THU

TNG QUAN MT S M HNH

NGHIN CU TC DNG CM MU

CA THUC TRN NG VT THC

NGHIM

KHA LUN TT NGHIP DC S

Ngi hng dn:

PGS.TS. o Th Vui

ThS. Trn Hng Linh

Ni thc hin:

B mn Dc Lc

Trng i hc Dc H Ni

H NI - 2014

LI CM N

Vi lng knh trng v bit n su sc ti xin gi li cm n chn thnh nht

ti

PGS.TS. o Th Vui B Mn Dc lc

ThS. Trn Hng Linh B Mn Dc lc

Nhng ngi thy trc tip hng dn, ch bo v gip ti trong sut qu

trnh lm kha lun.

Ti xin chn thnh cm n cc thy, c gio trng i hc Dc H Ni,

c bit cc thy c gio B mn Dc lc to mi iu kin ti thc hin tt

kha lun ny.

Ti cng xin gi li cm n ti Ban gim hiu, cc phng ban, cng ton th

cc thy c gio, cc cn b trng i hc Dc H Ni gip ti c th lnh hi

nhng kin thc qu gi v ngnh Dc trong sut nm nm hc.

Ti xin gi li cm n su sc ti gia nh, ngi thn, bn b lun ng

h, bn cnh ti trong qu trnh hc tp.

Cui cng, ti xin knh chc cc thy c lun mnh khe tip tc du dt

nhng th h hc tr mai sau.

H Ni, thng 5 nm 2013

Sinh vin

Phm Th Thu

Phm Th Thu

MC LC

T VN ............................................................................................................ 1

CHNG 1: TNG QUAN V QU TRNH CM MU V CC RI

LON QU TRNH CM MU ............................................................................ 3

1.1. Giai on co mch ti ch ............................................................................. 3

1.2. Giai on to nt tiu cu .............................................................................. 4

1.3. Giai on ng mu ....................................................................................... 8

1.4. Giai on co cc mu ng ......................................................................... 13

1.5. Giai on tan cc mu ng ........................................................................ 13

CHNG 2: PHNG PHP V QU TRNH TNG QUAN TI LIU .. 15

2.1. Ngun d liu .............................................................................................. 15

2.2. Chin lc tm kim .................................................................................... 15

2.2.1. D kin thc hin tng quan h thng .................................................. 15

2.2.2. nh hng li qu trnh tm kim ....................................................... 15

2.3. Quy trnh tm kim v la chn nghin cu ................................................ 16

2.3.1. Tm kim v tng hp cc m hnh gy chy mu bng tn thng ... 16

2.3.2. Tm kim v tng hp cc m hnh gy ri lon cm mu .................. 18

2.3.3. Tm kim b sung ................................................................................. 20

CHNG 3: TNG QUAN MT S M HNH NGHIN CU TC DNG

CM MU CA THUC TRN NG VT THC NGHIM .................... 21

3.1. M hnh gy chy mu bng tn thng. .................................................... 21

3.1.1. M hnh ct ui hoc chi..................................................................... 22

3.1.2. M hnh gy tn thng da, nim mc ................................................. 24

3.1.3. M hnh gy tn thng cc tng ......................................................... 26

3.1.4. M hnh gy chn thng no .............................................................. 30

3.1.5. M hnh gy tn thng cc mch mu ............................................... 34

3.2. M hnh gy ri lon thnh mch. ............................................................... 37

3.2.1. M hnh gy gin mch bng acetylcholin ........................................... 37

3.2.2. M hnh gy tng tnh thm thnh mch bng hp cht 48/80 ............ 39

3.3. M hnh gy ri lon tiu cu ...................................................................... 40

3.3.1. M hnh gim s lng tiu cu ........................................................... 40

3.3.2. M hnh gim chc nng tiu cu ......................................................... 43

3.4. M hnh gy ri lon cc yu t ng mu huyt tng ............................. 50

3.4.1. M hnh gim cc yu t ng mu huyt tng bng heparin. .......... 50

3.4.2. M hnh gim cc yu t ng mu bng gy thiu vitamin K ............ 52

3.4.3. M hnh gim cc yu t ng mu huyt tng bng gy bnh gan .. 56

3.4.4. M hnh gim cc yu t ng mu huyt tng bng di truyn ......... 59

CHNG 4: BN LUN V LA CHN CC M HNH NGHIN CU

.................................................................................................................................. .63

4.1. Bn lun v cc m hnh nghin cu ........................................................... 63

4.1.1. Cc yu t trong nghin cu nh hng n kt qu u ra ................ 63

4.1.2. M hnh chy mu bng tn thng ..................................................... 66

4.1.3. M hnh nghin cu trn cc giai on ca qu trnh cm mu ........... 68

4.1.4. Nhng im cn thiu ca ti .......................................................... 71

4.2. Bc u la chn m hnh nghin cu tc dng cm mu ca mt thuc 71

CHNG 5: KT LUN V XUT ............................................................. 75

5.1. Kt lun ........................................................................................................ 75

5.2. xut ......................................................................................................... 76

DANH MC CC K HIU, CH VIT TT

1 ADP Adrenosin diphosphat

2 aPTT Thi gian thromboplastin hot ha

(Activated Partial Thromboplastin)

3 DDAVP Desmopressin

(1-deamino-8-D-arginine vasopressin)

4 ELISA Xt nghim hp th min dch lin kt vi

enzyme

(Enzyme-linked immunosorbent assay)

5 FIX Yu t IX (Factor IX)

6 FVIII Yu t VIII (Factor VIII)

7 GP Glycoprotein

8 ICH Xut huyt no

(Intracerebral hemorrhage)

9 LMWH Heparin phn t lng thp

(Low-molecular-weight heparin)

11 PT Thi gian prothrombin

(Prothrombin Time)

12 TT Thi gian thrombin

(Thrombin Time)

13 UFH Heparin khng phn on

(Unfractionated heparin)

14 vWF Factor von-Willebrand

(Yu t von-Willebrand)

15 Ia, IIa, IIIa, IVa,

Va, VIIa, VIIIa,

IXa, Xa, XIa, XIIa,

XIIIa

(Tng ng )Yu t I, II, III, IV, V, VII, VIII,

IX, X, XI, XII, XIII hot ha

DANH MC CC BNG

STT Tn bng Trang

1 Bng 1.1. Cc yu t ng mu. 11

2 Bng 2.1. Cc m hnh trong nhm m hnh gy chy mu

bng tn thng

17

3 Bng 2.2. Cc bnh l d kin tm kim m hnh 18

4 Bng 2.3. Cc m hnh gy bnh tm kim c 20

2 Bng 3.1. im cc tiu ch hoi t v vim trn m bnh

hc cc tng.

30

3 Bng 3.2. Ch n AIN-76 thiu vitamin K. 55

4 Bng 3.3. im cho cc tn thng khp gi trn chut

hemophilia.

61

DANH MC CC HNH

STT Tn hnh Trang

1 Hnh 1.1. S kt dnh tiu cu thnh mch v tiu cu tiu

cu.

4

2 Hnh 1.2. Cc GP trn b mt tiu cu v cc cht hot ha

tiu cu.

6

3 Hnh 1.3. S hnh thnh Thromboxan A2. 7

4 Hnh 1.4. S ng mu. 9

9 Hnh 2.1. V tr ct trong m hnh ct ct chi 23

5 Hnh 2.2. V tr ca ng rch trn m hnh gy tn thng

gan.

27

6 Hnh 2.3. Gy tn thng gan theo phng php m thng. 28

7 Hnh 2.4. V tr m kim v ng knh vt thng trn tnh

mch i

35

10 Hnh 2.6. Vt knh trn da chut quan st cc mch mu

di knh hin vi.

38

11 Hnh 4.1. S cc hng la chn m hnh nghin cu. 74

1

T VN

Chy mu l triu chng xy ra khi c chn thng, tai nn, phu thut v

trong cc bnh di truyn nh hemophilia hoc von-Willebrand, cng nh cc bnh

mc phi khc (ri lon thnh mch, ri lon s lng v chc nng tiu cu, ri

lon cc yu t ng mu, v ri lon qu trnh tiu fibrin) [15], [70], [98]. Mt

khc, hin nay s lng ngi s dng thuc chng huyt khi d phng nguy

c tim mch khng ngng tng. Do , chy mu do qu liu thuc chng ng xy

ra thng xuyn hn v nghim trng hn vi cc triu chng c th nh nh chy

mu di da, cc chm di da, chy mu cam cho n cc triu chng nng

xut huyt t mu no.

Qua nhiu nghin cu, qu trnh cm mu c hiu bit su hn, nh

gi ra nhng hng mi trong nghin cu v pht trin cc thuc cm mu mi.

Hin nay, c nhiu thuc cm mu c s dng nh: cc thuc cm mu ti ch

nh keo fibrin, thrombin, zeolite hoc cc thuc cm mu ton thn nh phc hp

yu t VIII, thrombin) c c, thuc desmopressin, thuc chng tiu fibrin (acid

tranexamic). Trong , cc phc hp yu t c c l c s dng ph bin hn

c. Nhng cc thuc ny cha c tc dng cm mu, c bit trong cc trng

hp mu chy nhiu, thuc cng gy nhiu tc dng khng mong mun: truyn

nhim virus, phn ng min dch, cc thuc cm mu ti ch gy bm dnh v vim

ti v tr b tn thng [54], [75], [79], [94].

V nhng nguyn nhn trn nn vic tm kim, nghin cu cc thuc cm

mu mi c hiu qu tt, t tc dng khng mong mun, l yu cu cn thit v cp

bch. T lc pht hin ra kh nng cm mu ca mt dc liu hoc mt hp cht

ha dc n lc bin n tr thnh mt ch phm thuc hon chnh a vo s

dng trn lm sng l c mt chng ng di, trong , giai on tin lm sng

th tc dng dc l l khu rt quan trng. thc hin cc nghin cu , khng

th thiu cc m hnh thc nghim ph hp gip chng minh thuc thc s c

tc dng hay khng.

2

Hin nay, ti Vit Nam, cc m hnh nghin cu tc dng cm mu ca

thuc cn rt t v hn ch, cha c thng k mt cch y . Nhm a ra mt

ci nhn tng hp cc m hnh nghin cu cc thuc cm mu c thc hin

trn th gii, cng nh so snh, nh gi v nhn nh cc m hnh c kh nng

thc hin Vit Nam, phn tch cc hng nghin cu v cc m hnh cn s dng

nghin cu v tc dng cm mu ca thuc, chng ti tin hnh thc hin ti

Tng quan mt s m hnh nghin cu tc dng cm mu ca thuc trn ng

vt thc nghim vi mc tiu:

Tng quan v qu trnh cm mu v cc ri lon cm mu.

Tng quan v mt s m hnh nghin cu tc dng cm mu ca thuc.

Bn lun cc m hnh v bc u nh hng la chn m hnh nghin cu

tc dng cm mu ca thuc.

3

CHNG 1: TNG QUAN V QU TRNH CM MU V CC RI

LON QU TRNH CM MU

phc v cho qu trnh tm kim, chng ti tin hnh tng quan ngn gn

v qu trnh cm mu (v cc yu t lin quan n vic gy m hnh) cng cc bnh

l ri lon qu trnh cm mu nh sau:

Cm mu l mt qu trnh din ra nhm hn ch hoc ngn cn mu chy ra

khi thnh mch khi thnh mch b tn thng. Qu trnh cm mu c thc hin

qua 5 giai on: co mch, to nt tiu cu, ng mu, to cc mu ng v tan cc

mu ng [3], [10].

1.1. Giai on co mch ti ch

Co mch ti ch l phn x u tin ca c th khi mch mu b tn thng,

s co mch s hn ch lng mu thot ra ngoi. Co mch cn c tc dng lm tc

lu chuyn mu chm li, to iu kin cho vic hnh thnh nt tiu cu v cc

mu ng. Co mch xy ra theo hai c ch: c ch thn kinh l do nhng xung ng

au ni b tn thng s hot ha thn kinh giao cm gy phn x co mch. C ch

th dch l do t bo ni mc gii phng cc cht angiotensin II v tiu cu bi tit

serotonin v thromboxan A2 u c tc dng co mch. Cc phn x trn lm mch

mu co li, ng knh thu nh, ngn mu chy ti vt thng.

Hiu qu co mch c ngha trong vic to inh cm mu ban u, tuy nhin

ch c ch co mch thi l cha cm mu m cn thit c cc c ch khc

tham gia vo. Mt khc, vi cng mt tn thng, hiu qu co mch cc mch

mu ln km hn nhiu so vi cc mch mu nh, nh vy, khi nghin cu v tc

dng co mch ca thuc cm mu cn lu k iu ny.

Giai on co mch c s tham gia ca nhiu yu t, trong , vai tr quan

trng nht thuc v lp t bo ni mc. Khi c tn thng thnh mch mu, t bo

ni mc tit ra cht co mch cc b l angiotensin II. Angiotensin II l mt cht c

tc dng co mch mnh. Song song vi vai tr cm mu, t bo ni mc cng c vai

tr quan trng trong vic chng huyt khi (bi tit NO, prostacyclin I2), nh vy

m mu lu thng tt trong thnh mch, ch khi c tn thng th qu trnh cm

mu mi c hot ha [3], [10].

4

Trong cc bnh l chy mu do thnh mch, tnh trng mu vn bnh thng

nhng cc mch mu b tn thng lm thay i tnh thm thnh mch v trng

lc mch mu [79]. Cc thay i trn lm thnh mch khng thc hin c trn

vn vai tr ca n trong giai on co mch, dn n xut huyt trn cc vng nim

mc ming, mi..., hoc xut huyt trn da trong nhng tn thng d rt nh.

Trong cc bnh l ny, thi gian chy mu ko di khi s lng tiu cu vn bnh

thng. Thnh mch b tn thng do cc nguyn nhn:

Vim do nhim khun, d ng, c t, min dch: lm tng sc thm thu ca

thnh mch mu, gy thot mch, ph n v d b xut huyt.

Di truyn lm gin cc mao mch trn cc bnh nhn Rendu Osler-Weber.

1.2. Giai on to nt tiu cu

Qu trnh to nt tiu cu ti v tr vt thng: [5], [3], [10]

Khi thnh mch b tn thng, lp t bo ni mc b ph v, cc t chc

di ni mc nh collagen, mng nn, vi si, cht chun... c bc l. y l iu

kin c bn cho hin tng dnh v ngng tp xy ra, trc ht l tiu cu trong

phn th tch mu ti vng c tn thng.

Hnh 1.1. S kt dnh tiu cu thnh mch v tiu cu tiu cu.

S kt dnh ca tiu cu vo collagen di lp t bo ni mc xy ra gn nh

ngay lp tc. Lp collagen tch in dng, tiu cu tch in m v c receptor vi

collagen, do , tiu cu d dng kt dnh vi thnh mch tn thng. Bn thn t

bo ni m b thng cn gii phng ra yu t von-Willebrand gip gn cc phn t

glycoprotein (GP) trn mng tiu cu vo collagen ca t bo ni mc (nh hnh

1.1).

5

Sau khi kt dnh vo ni c tn thng, tiu cu c hot ha, b mt tr

nn x x, ng thi gii phng cc cht: ADP, serotonin, fibronectin lm tng Ca2+

trong tiu cu, dn n hot ha GP IIb/IIIa trn mng tiu cu, lm tiu cu khc

lu ng trong mu kt tp vi tiu cu va b kt dnh (hnh 1.1). Cc tiu cu mi

kt t s tip tc c hot ha v bi tit cc cht ha hc lm cho cng c thm

nhiu tiu cu n kt t hnh thnh nt tiu cu. Cc tiu cu sau khi hot ha d

dng dnh vi fibrin (c to thnh giai on ng mu), to cc mu ng bn

vng.

S hnh thnh nt tiu cu c th s b bt kn tn thng lm cho mu

ngng chy nu tn thng mch mu nh. Thi gian hnh thnh nt tiu cu

(thi gian mu chy) bnh thng khong 2 4 pht (tnh t khi mu chy ra khi

nhng mch nh cho n lc mu ngng chy).

Giai on to nt tiu cu l giai on c s tng tc ca cc yu t thuc

thnh mch v yu t tiu cu, trong , tiu cu l yu t quan trng v cn thit

nht. Tham gia vo qu trnh cm mu, tiu cu c 3 chc nng chnh: kt dnh

(dnh tiu cu vi collagen v cc thnh phn khc ca t chc di ni mc), kt

tp (kh nng kt dnh ln nhau), thay i hnh dng v gii phng ht (qu trnh

tiu cu phng to ln, hnh thnh chn gi,co rt v gii phng cc ht cha trong

tiu cu: serotonin, yu t 3 tiu cu, ADP, Ca2+...). thc hin c 3 chc nng

trn, tiu cu c cu trc rt c bit: t bo khng nhn c cu to bi hai thnh

phn chnh: [3]

Mng tiu cu tch in m rt mnh, c nhiu ch lm vo trong bo tng

lm tiu cu c tnh xp v tng din tch b mt ln rt nhiu. Trn mng tiu cu

c cha cc phn t GPIb, GPIIb/IIIa, cc receptor ca cc yu t collagen,

thrombin, ADP... Nh h thng cc GP cng nh cc receptor ny m khi thnh

mch tn thng bc l lp collagen, tiu cu s d dng kt dnh vo khi ng

qu trnh hnh thnh nt tiu cu.

Bo tng ca tiu cu c actinin, myosin v thrombosthenin. Cc protein

ny gip tiu cu c hot ha c th co li v gii phng cc cht ng bn

6

trong cc ht bo tng nh: Ca2+, serotonin, adenosinediphosphat (ADP), ... chng

c gi l cht kch hot tiu cu.

C ch to nt tiu cu c to nn bi s ng gp ca mt s i phn t,

chng c vai tr nh cht dnh hay cht hot ha tiu cu. Chng bao gm cc GP

trn b mt tiu cu, cc ADP, von Willebrand v thrombin: [3], [10]

GP Ib v GP IIb/IIIa: l cc protein c phn b u trn mng bo tng

ca tiu cu. Khi tiu cu c hot ha, mng tiu cu dch chuyn v phn t cc

GP c bc l. Cc protein huyt tng nh fibrinogen s ni nhng GP IIb/IIIa

ca cc tiu cu vi nhau, to nn s kt tp tiu cu. Bn cnh , cc GP khc

cng ni vi cc yu t von-Willebrand, collagen gy dnh tiu cu vi ni mc

nh hnh 1.2.

Yu t von-Willerbrand (v-WF): l mt glycoprotein c tng hp t t bo

ni mc v mu tiu cu, lu hnh trong huyt tng. v-WF c vai tr to ln trong

cm mu k u, l cht keo sinh hc gn cc phn t GPIb v GPIIa trn mng

tiu cu vi collagen, dnh tiu cu vi t chc di ni mc, to nn inh cm

mu. Ngoi ra, khi lu hnh trong mu, v-WF gn vi yu t VIII di dng phc

hp VIII - v-WF (l mt protein mang yu t VIII). Do vy, mt s bnh l lm

gim vWF th cng lm gim yu t VIII, ko di thi gian chy mu.

Hnh 1.2. Cc GP trn b mt tiu cu v cc cht hot ha tiu cu

(Ngun: http://www.jhoonline.org/content/4/1/44/figure/F1)

Thromboxan A2: hot ha receptor GP IIa/IIb trn mng tiu cu gy kt tp

tiu cu [5]. Thromboxan A2 c tng hp khi tiu cu hot ha, cc enzym nh

7

cyclooxygenase v thromboxan synthetase xc tc chuyn acid arachidonic thnh

thromboxan A2. V sau, do lng ADP v thrombin ngy cng nhiu, hot ha

cng nhiu enzym phospholipase chuyn phospholipid thnh acid arachidonic,

lm cho lng thromboxan A2 c tng hp ra li cng nhiu hn na.

Hnh 1.3. S hnh thnh thromboxan A2 [5]

ADP: l mt cht gy ngng tp tiu cu rt mnh c trong bo tng tiu

cu. Khi tiu cu kt dnh vo collagen v vi si, n s thay i hnh dng v gii

phng ra ADP (chnh l yu t XIII ca tiu cu). Mt lng ADP nh khc c

to thnh do hng cu b v hoc do s thoi ha ATP thnh ADP di tc ng

ca thrombin. V sau s kt tp tiu cu cng tng th lng ADP cng c phng

thch ra nhiu hn. C ch gy ngng tp tiu cu ca ADP l: ADP gn vo

receptor ca n trn mng tiu cu, gy hot ha receptor GP IIb/IIIa, ADP cng

thc y qu trnh to ra thromboxan A2.

Tiu cu c mt vai tr quan trng trong ng mu, do , ri lon tiu cu

k c v s lng hay chc nng u c th gy chy mu [6]. Gim s lng tiu

cu c th l do c ch qu trnh sinh tiu cu ty xng (chiu x, thuc, cc u c

tnh), hoc do tng hy tiu cu trong l lch (thuc, nhim trng do vi khun hoc

virus, lupus ban ). Trong cc bnh l ny, cc xt nghim ng mu (aPTT,

TT, PT) l bnh thng, ch c s lng tiu cu gim lm thi gian chy mu ko

di.

Gim chc nng tiu cu khi tiu cu thiu ht mt trong ba chc nng ca

n. Cc phn t c vai tr nh cht dnh hay cht hot ha tiu cu b thiu ht

(trong cc bnh di truyn) hoc b c ch (do dng thuc khng tiu cu) l nguyn

8

nhn gy chy mu ko di. Thiu ht cc GPIIb/IIIa trn tiu cu trong bnh

Thrombasthenia Glanzmann, thiu ht yu t von-Willebrand gp bnh von-

Willebrand, gim cc ht lu gi trong tiu cu gp hi chng Hermansly

Pudlak. Bn cnh , cc thuc khng tiu cu nh aspirin (c ch hnh thnh

thromboxane A2), clopidogrel (c ch ADP hot ha tiu cu) u c kh nng lm

gim chc nng tiu cu. Trn lm sng, thng gp cc biu hin nh ca bnh

(nh chy mu nim mc, chy mu ko di khi c tn thng, thm ch l khng

c triu chng), nhng c th tin trin nng gy ra cc trng hp xut huyt no.

Xt nghim cho thy thi gian chy mu ko di trong khi s lng tiu cu v thi

gian ng mu vn bnh thng, xt nghim o dnh v o kt tp tiu cu u

cho gi tr gim [1], [6], [15].

1.3. Giai on ng mu

Qu trnh ng mu: [3], [10]

ng mu l mt chui cc phn ng ha hc ca cc yu t ng mu c

trong huyt tng, cc m tn thng v tiu cu. Khi mt yu t c hot ha, n

s ng vai tr enzym xc tc cho s hot ha yu t tip theo, do to thnh mt

dy chuyn phn ng dn n s hnh thnh mng li fibrin v lm mu ng li.

Kt qu l mu chuyn t th lng thnh th c, do s chuyn fibrinogen thnh

fibrin khng ha tan v cc si fibrin ny s trng hp to ra mng li fibrin giam

gi hng cu v lm mu ng li. Qu trnh ny din ra qua 3 giai on v c th

c tm tt nh hnh 1.4:

Giai on 1: giai on to prothrombinase.

Khi mch mu b tn thng, ng mu c khi ng ng thi bi c 2

con ng:

Con ng ng mu ngoi sinh din ra rt nhanh, bt u bng s gii

phng tromboplastin ca m (yu t III) t mch mu hoc m b tn

thng. Yu t III s kt hp v hot ha yu t VII. Phc hp yu t IIIa

VIIa v ion Ca++ s hot ha yu t X. S kt hp ca yu t Xa vi

9

phospholipid tiu cu, ion Ca++ v yu t Va to thnh phc hp

prothrombinase (hnh 1.4).

Hnh 1.4. S ng mu [3]

Con ng ni sinh: c hot ha khi bn thn mu b tn thng hoc tip

xc vi b mt l (v d nh thnh ng nghim). Yu t ng mu u tin

c hot ha l yu t XII (yu t tip xc). Sau , cc yu t XI, IX s

c hot ha theo kiu dy chuyn. S hot ha yu t X trong c ch ni

sinh cn s c mt ca phospholipid c gii phng t tiu cu, ion Ca++ v

yu t VIII. Sau khi hnh thnh yu t X hot ha, s to thnh phc hp

prothrombinase xy ra tng t nh c ch ngoi sinh.

C hai con ng ng mu ni sinh v ngoi sinh u to thnh

prothrombinase. y l enzym xc tc cho s chuyn prothrombin thnh thrombin,

10

qua hnh thnh nn mng fibrin lm mu ng li. Giai on to prothrombinase

cng l giai on phc tp nht trong qu trnh ng mu.

Giai on 2: giai on chuyn prothrombin thnh thrombin

Phc hp enzym prothrombinase c to thnh giai on 1 xc tc cho

phn ng chuyn prothrombin thnh thrombin. Thrombin (yu t IIa) xc tc cho

phn ng chuyn fibrinogen thnh fibrin, v vy n ng vai tr i vi s hnh

thnh v n nh mng fibrin. Ngoi ra, n cn ng vai tr xc tc cho s hot ha

ca yu t XIII, V, VII, XI m rng hot ng ng mu. Mt khc, thrombin

cng c tc dng gii hn s lan rng ca qu trnh ng mu bng vic hn ch

hot ng ca chnh n thng qua hot ha protein C. Do vy, vi mt lng nh

thrombin c sn trong mu v sau c sinh ra nhanh chng theo con ng

ngoi sinh s lp tc to ra mt feedback dng tnh to nhiu thrombin hn,

hot ha qu trnh hnh thnh cc mu ng.

Giai on 3: giai on chuyn fibrinogen thnh fibrin

Di tc dng ca thrombin cc phn t fibrinogen bnh thng ha tan s

chuyn thnh fibrin n phn. Cc fibrin n phn s trng hp to ra si fibrin,

di tc dng ca yu t n nh fibrin yu t XIIIh, lin kt gia cc si fibrin

bn hn, mng fibrin tr nn bn vng.

Sau khi to c mng fibrin giam gi tiu cu v cc thnh phn khc ca

mu, qu trnh ng mu cng c m rng hn na bi yu t 3 tiu cu (do tiu

cu bin dng gii phng ra t giai on to nt tiu cu), lm dnh v ngng tp

tiu cu nhiu hn na. Khi dnh v ngng tp tiu cu nhiu hn th phng thch

li cng mnh m. Vng xon ny mi lc mt m rng. Tiu cu theo dng mu s

nhanh chng b thu ht vo vng xon , cng v sau cng dnh nhiu hn bi l

chnh s dnh v thay i hnh dng ca n lm cho hin tng dnh tng ln na.

Cc yu t tham gia vo qu trnh ng mu hu ht c tng hp gan v

gii phng vo huyt tng di dng tin cht khng hot ng, ch khi no c

hot ha th chng mi tham gia vo qu trnh ng mu. Cc yu t ng mu

dng tin cht c tm tt trong bng:

11

Bng 1.1. Cc yu t ng mu [3]

Danh php quc

t

Tn thng thng Ni khu tr Ni sn xut

Yu t I Fibrinogen Huyt tng T bo gan

Yu t II Prothrombin Huyt tng T bo gan

Yu t III Tromboplastin ca m M M

Yu t IV Ion calci Huyt tng

Yu t V Proaccelerin

Yu t khng bn

Huyt tng T bo gan

Yu t VII Proconvertin

Yu t bn vng

Huyt tng T bo gan

Yu t VIII Yu t chng hemophilia A Huyt tng T bo gan

Yu t IX Yu t Chrismast

Yu t chng hemophilia B

Huyt tng T bo gan

Yu t X Yu t Stuart

Yu t Stuart Prower

Huyt tng T bo gan

Yu t XI Tin tromboplastin huyt

tng

Yu t chng hemophilia C

Huyt tng T bo gan

Yu t XII Yu t Hageman

Yu t tip xc

Huyt tng T bo gan

Yu t XIII Yu t n nh Fibrin Huyt tng T bo gan

Yu t tiu

cu

Yu t III ca tiu cu Tiu cu T bo gan

Trong , cc yu t cn lu l: [3], [10]

Nhm cc yu t ph thuc vitamin K

Gm cc yu t II, VII, IX, X. c im chung l u cn s c mt ca

vitamin K chuyn thnh dng hot ng (phn ct mt hoc hai dy ni peptid).

12

Cc yu t trong nhm ny c lu tr n nh trong huyt tng v khng b tiu

th trong qu trnh ng mu (tr yu t II). Khi c th thiu vitamin K th gan ch

tng hp ra tin cht ca cc yu t trn, cc tin cht c hot tnh sinh hc ng

mu rt thp v i khi cn c hot tnh c ch ng mu. Khi thiu cc yu t ny,

bnh nhn d b xut huyt, cc xt nghim theo xu hng gim ng: PT ko di,

nh lng nng cc yu t trong huyt tng gim.

Anti-thrombin III (AT III): bt hot hu ht cc yu t ng mu theo con

ng ni sinh (XIIa, XIa, IXa, Xa, thrombin) v c plasmin. C ch bt hot l AT

III to ra mt phc hp bn vng, khng hi phc vi cc yu t ng mu hot

ha, thng qua cc v tr gn ca n trn b mt cc yu t ng mu ny. Cc yu

t sau khi b bt hot s b loi ra khi tun hon. Tc dng ca heparin din ra

chm, tuy nhin, khi c mt heparin, tc dng ca AT III tng ln 1000 ln. AT III

gi mt vai tr sinh l rt quan trng, khi b thiu s gia tng nguy c tc mch do

huyt khi.

Yu t VIII: l mt protein ng mu lu hnh trong mu, sau khi tng hp

ra mt thi gian ngn (8-12 pht), chng s gn vi yu t von Willebrand nh

m m bo c tnh n nh ca yu t ny. Thiu ht yu t VIII gp nhiu nht

trong bnh hemophilia A, gy chy mu t pht cc bnh nhn mc bnh nng

(nng yu t VIII di mc < 5% mc bnh thng). Yu t VIII b gim trong

huyt tng ca bnh nhn hemophilia A v bnh von-Willebrand.

Khi thiu ht mt hoc nhiu yu t ng mu s lm bt thng cc xt

nghim ng mu v gy chy mu ko di. Thiu ht mt yu t ng mu gp

trong cc bnh Hemophila A (thiu ht yu t VIII do t bin gen), Hemophilia B

(thiu ht yu t IX, bnh Christmas), hoc mt s bnh di truyn khc. Thiu ht

nhiu yu t ng mu gp cc bnh nhn s dng heparin, thiu vitamin K (do

khng hp thu hoc do dng thuc khng vitamin K), hoc bnh l gan. Cc bnh

l ny lm cc yu t ng mu gim tng hp hoc tng hp bnh thng nhng

khng hot ha c, do , khng tham gia vo qu trnh ng mu mt cch y

, mng fibrin khng hon ton ph kn vt thng cm mu [1], [6], [15].

13

Cc xt nghim giai on ny, ngoi thi gian chy mu ko di, cn c ba

xt nghim c trng cho tng con ng ng mu l: [2], [10]

Thi gian prothrombin (PT): xt nghim dng nh gi con ng ng mu

ngoi sinh (thm d cc yu t I, II, III, V, VII, X). Do , dng khng

vitamin K, bnh Hemophilia lm thi gian prothrombin ko di.

Thi gian thromboplastin tng phn hot ha (aPTT): xt nghim nh gi

thi gian ng mu ni sinh (cc yu t I, II, V, VIII, IX, X, XI, XII). aPTT

ko di trong trng hp s dng chng ng heparin.

Thi gian thrombin (TT): xt nghim thm d giai on sau cng ca qu

trnh ng mu, giai on chuyn fibrinogen thnh fibrin, TT ko di khi s

dng heparin hoc khi c tnh trng tng fibrinogen mu. Trong cc bnh l

v gan th c ba ch s PT, aPTT, TT u ko di.

1.4. Giai on co cc mu ng

Sau khi mu ng khong t 1- 2 gi, cc mu ng co li v gii phng ra

ton b dch ca n. Hin tng co cc mu ng c tc dng lm mp vt thng

khp li gn nhau hn to iu kin cho s ha so. Ngi ta cho rng, tc dng

co ny l ca thrombostenin, mt protein c gii phng ra t tiu cu, tc dng co

ca thrombostenin tng t nh actomysin ca c.

Khi s lng hoc cht lng tiu cu gim s lm cho thi gian co cc mu

ko di hn v cc mu ng co khng hon ton. S co cc mu ng c hot

ha bi thrombin v ion Ca++. Do vy, thiu ion Ca++ cng lm cc mu ng co

khng hon ton.

1.5. Giai on tan cc mu ng

L hin tng cc mu ng tan ra di tc dng ca plasmin. Plasmin l

mt enzym c hot ha t plasminogen, c kh nng phn hy fibrin mnh,

chuyn fibrin khng ha tan thnh cc sn phm thoi ha c trng lng phn t

thp hn v ha tan trong huyt tng, do , lm tan cc mu ng [10]. Bnh

thng plasmin khng c trong mu lu thng, khi fibrin ca cc mu ng xut

hin, ngay lp tc xy ra hin tng kch hot plasminogen. Tan cc mu ng l

14

mt trong nhng c ch chng ng ca chnh c th khi c xut hin cc mu

ng ngn nga tc mch v to iu kin cho qu trnh lin so, tuy nhin, khi

qu trnh hnh thnh plasmin khng c kim sot k cng s gy ra ri lon tiu

si huyt. Nguyn nhn c th do thiu ht cht c ch hot ha plasminogen, thiu

ht yu t XIII. Cc ri lon ny l tng hy fibrin nn lm tan cc mu ng nhanh

chng, gy chy mu trn lm sng v xt nghim cho thy thi gian tan cc mu

ng ko di [1], [6], [15].

15

CHNG 2: PHNG PHP V QU TRNH TNG QUAN TI LIU

2.1. Ngun d liu

Tin hnh tm kim trong c s d liu Pubmed (bao gm tt c ti liu c t

trc n nay, khng gii hn thi gian) thu thp cc nghin cu ph hp.

Thi gian tm kim: t 1/12/2013 15/4/2014

2.2. Chin lc tm kim

2.2.1. D kin thc hin tng quan h thng

S dng t kha ban u hemostatic model, thng qua mc y khoa

MeSH, chng ti thu c danh mc cc t ng ngha: hemostatic assay,

antihemorrhagic model, antihemorrhagic assay v b sung cc dng s nhiu

ca n l hemostatic models, hemostatic assays, antihemorrhagic models,

antihemorrhagic assays.

Cc cm cha c t trong ngoc kp v tt c cc cm t trn c kt

hp bi ton t OR to thnh c php cui cng, g vo tm kim ca Pubmed.

Chng ti s dng chc nng gii hn duy nht l Other animals (nghin

cu trn ng vt) thu c cc m hnh trn ng vt thc nghim c lin quan.

Sau khi thc hin lnh tm kim th s kt qu thu c l 23806 kt qu

(trc 12/2013). Vi s lng kt qu ny th vic tin hnh mt tng quan h

thng trong phm vi kh nng ca chng ti l khng th.

2.2.2. nh hng li qu trnh tm kim

Do vic thc hin mt tng quan h thng l khng thc hin c, do ,

chng ti ra sot li cc thng tin v qu trnh cm mu, bnh l cc qu trnh ny

cng nh cc thng tin t sch dc l thc nghim c bn Drugs discovery and

evaluation: pharmacology assay. Theo , qu trnh tm kim chia lm 2 phn nh

sau:

Phn mt: tin hnh tm kim v tng hp cc m hnh gy chy mu bng tn

thng.

Phn hai: tin hnh tm kim v tng hp cc m hnh gy chy mu bng cch

gy cc ri lon cm mu.

16

2.3. Quy trnh tm kim v la chn nghin cu

2.3.1. Tm kim v tng hp cc m hnh gy chy mu bng tn thng

S dng t kha bleeding model v dng s nhiu bleeding models trn

tm kim ca th vin y khoa Pubmed, s dng t ni l ton t OR, vi chc nng

gii hn duy nht l other animals. Sau khi thc hin lnh tm kim th s kt qu

thu c l 99 kt qu.

La chn cc nghin cu: c k tiu v tm tt ( abstract) ca tt c cc

nghin cu. Tt c nhng bi bo ny u c c bn y da trn kh nng

cho php ca ngun thng tin m nhm nghin cu c th ly c. Cc kt qu

c trch xut mt cch ngn gn vo bng thun tin cho vic gp cc bi bo

ging nhau.

Tiu chun la chn v loi tr

Tiu chun la chn: c la chn, cc nghin cu phi tha mn nhng

tiu chun sau:

+ Loi hnh nghin cu: invivo

+ Thi gian xut bn: ln tm kim th nht trc 12/2013 v ln tm kim th

2 trc 15/4/2014.

+ ng vt nghin cu: chut nht (mouse, mice), chut cng (rat), th

(rabbit), ch (dog).

Tiu chun loi tr

Tt c nhng nghin cu in vitro, cc nghin cu trn cc ng vt khc,

hoc nhng nghin cu khng trnh by phng php c th tin hnh gy m

hnh u b loi b.

Tm kim b sung:

B sung cc ti liu tham kho ca mt s bi bo chn, nhm tm kim

cch gy m hnh c th, cc k thut c cp v tn m cha c trong cc bi

bo chn. B sung mt s ti liu nh gi, bn lun v cc m hnh trn.

Cc m hnh tm kim c:

17

Qua qu trnh sng lc thng tin ban u, chng ti nhn thy: cc m hnh

gy tn thng m chng ti tm kim c c cc thng s nh gi l tng t

nhau (o thi gian mu chy v lng mu mt, cng mt s thng s khc c

trng cho mi m hnh). Do , chng ti chia 21 m hnh tm kim c vo 5

m hnh ln nh sau:

Bng 2.1. Cc m hnh trong nhm m hnh gy chy mu bng tn thng

1 M hnh ct ui hoc

chi:

+ Gy chy mu bng ct ui chut

+ Gy chy mu bng ct ui ch

+ Gy chy mu bng ct ui ch

2 M hnh gy t thng

da v nim mc

+ Gy tn thng nim mc mi th

+ Gy tn thng nim mc li ch

+ Gy tn thng trn tai th

+ Gy tn thng da chut

+ Gy tn thng m mm

3 M hnh gy tn thng

cc tng

+ Gy tn thng gan trn chut

+ Gy tn thng gan trn th

+ Gy tn thng lch

+ Gy tn thng thn

4 M hnh gy chn

thng no

+ Gy tn thng no th

+ Gy chn thng no chut

+ Gy xut huyt ni s (ICH) bng

collagenase

5 M hnh gy tn thng

cc mch mu

+ Gy tn thng tnh mch ch di

th

+ Gy tn thng tnh mch cnh th

+ Gy tn thng tnh mch i chut

+ Gy tn thng ng mch ch bng

chut

18

+ Gy tn thng ng mch i chut

+ Gy tn thng ng mch mc treo

chut

2.3.2. Tm kim v tng hp cc m hnh gy ri lon cm mu

Chng ti tng hp cc m hnh gy bnh (gy ri lon cm mu) theo

qu trnh nh sau:

Bc 1: D kin cc bnh l cn tm kim m hnh

Khi qut cc bnh l ri lon cm mu thng gp trn lm sng v tm

kim cc m hnh cho cc bnh l . Bnh l c d kin l cc bnh l thng

gp trn lm sng, trn 3 yu t chnh; thnh mch tiu cu cc yu t ng

mu.

Sau khi khi qut v r sot trong cc sch bnh l th cc bnh d kin tm

kim nh sau

Bng 2.2. Cc bnh l d kin tm kim m hnh

Bnh l T kha

Thnh

mch

Di truyn, bnh l gy vim, gy

ra tnh trng gin mch hoc

tng tnh thm thnh mch

Gin mch

(vasodilatation)

Tnh thm permeability

Tiu cu Bnh l lm gim s lng tiu

cu

Thrombocytopenia

Bnh l lm gim cht lng

(hoc chc nng) tiu cu

Platelet function

disorders,

Thrombocytopathy

Cc yu t

ng mu

Bnh l lm ri lon con ng

ng mu ni sinh (dng chng

ng heparin)

Heparine

Bnh l lm ri lon con ng

ng mu ngoi sinh (thiu

vitamin k deficiency,

19

vitamin K do dng thuc chng

ng hoc do n ung)

Bnh l lm ri lon con ng

ng mu chung (bnh gan)

hepatic disease, liver

disease

Bnh l lm gim mt yu t

ng mu nht nh (di truyn)

hemophilia

Bc 2: Tm kim

S dng t kha l t bnh, hoc cc tc nhn gy bnh tm kim. Cc t

kha kt hp bao gm: hemostatic model OR hemostatic models. Bnh l

trn thnh mch, kt hp vi cm: vascular model OR vascular modelsOR

vessel model OR vessel models. Vi cc bnh l trn tiu cu, kt hp t kha

tm kim l: platelet model OR platelet models, v bnh l trn cc yu t ng

mu kt hp vi cm: coagulation model OR coagulation models. G cc t

kha vo tm kim trn th vin y khoa Pubmed.

Cc nghin cu c la chn phi tha mn: l cc nghin cu in vivo, c

qu trnh gy bnh l r rng.

Bc 3: Tng hp kt qu

Cc nghin cu c cch gy m hnh tng t nhau c gp vo mt m

hnh chung, cc thng s cn o lng c tng hp t tt c cc bi bo cp

n m hnh .

Chng ti cng b sung cc tc nhn xut hin trong cc m hnh gy chy

mu bng tn thng mc 2.3.1 trn. Km theo , l cc ti liu tham kho ca

cc bi bo la chn tng hp, nhm m t chnh xc qu trnh gy bnh l v

cch nh gi cc thng s, phc v tt nht c th cho c l thc nghim..

Kt qu nh sau:

20

Bng 2.3. Cc m hnh gy bnh tm kim c

M hnh

S lng m

hnh

M hnh gy ri lon

thnh mch:

+ Gy gin mch 1

+ Gy tng thm 1

M hnh gy ri lon

tiu cu

+ Gy gim s lng tiu cu 2

+ Gy gim cht lng tiu cu 3

M hnh gy ri lon

cc yu t ng mu

+ Gy bng heparin 1

+ Gy thiu vitamin K 2

+ Gy bnh gan 2

+ Gy hemophilia 2

2.3.3. Tm kim b sung

Trong qu trnh tm kim, chng ti cng nhn thy c mt thuc c tn l

Ankaferd blood stopper (ABS), y l chit xut hn hp 5 dc liu dn gian

ca Th Nh K. Tin hnh tm kim t kha chnh l tn thuc ny trn Pubmed,

chng ti nhn thy, y l mt ch phm thuc c bt u nghin cu t 1/2008,

t mt dc liu c tim nng thnh mt thuc c dng trn lm sng. S lng

kt qu tm kim c l 128 bi bo, sau khi s dng chc nng gii hn (lc)

other animals th kt qu chng ti tm kim c l 32 kt qu. c tt c 32 bi

bo ny: bn y (full text) trong kh nng ca nhm nghin cu v tt c phn

tm tt (abstract)), sau , kt hp tt c cc nghin cu m chng ti tm kim

c s b a ra nh hng nghin cu cho mt thuc cm mu.

21

CHNG 3: TNG QUAN MT S M HNH NGHIN CU TC DNG

CM MU CA THUC TRN NG VT THC NGHIM

Chy mu l trng thi xy ra khi c cc tn thng ln cc c quan, mch mu

trn c th, lm mt tnh ton vn ca mch mu. Trn cc bnh nhn c sn cc ri

lon cm mu th d dng xy ra chy mu qu mc ch vi mt va p nh hay tn

thng nh, thm ch c th c chy mu t pht [6], [10], [98]. Da trn nhng

nguyn tc , cng vi nhng hiu bit v qu trnh cm mu v cc bnh ri lon

chy mu, ngi ta to ra cc m hnh chy mu thc nghim dng nghin cu

tc dng cm mu ca thuc. Cc m hnh hin nay hay c s dng c th chia

lm 5 loi:

M hnh gy chy mu bng tn thng

M hnh gy ri lon thnh mch

M hnh gy ri lon tiu cu

M hnh gy ri lon cc yu t ng mu

M hnh gy ri lon tiu fibrin

Cc m hnh ny c th tin hnh trn ng vt bnh thng hay ng vt c bnh

l v ri lon qu trnh cm mu, tuy nhin, chy mu t pht l rt him khi xy ra,

do vy, trong tt c cc m hnh, gy tn thng l phng php duy nht kim

tra hot ng cm mu ca c th.

3.1. M hnh gy chy mu bng tn thng.

m phng cc tnh trng chy mu do chn thng, tai nn, phu thut,

gy ra chy mu tng, no, mch mu..., th trn thc nghim c th gy chy mu

bng cch gy tn thng cc c quan tng, no, cc mch mu, trn da, ct ui

hoc chi ca con vt tng ng. Cc tn thng ny u c tiu chun ha v

thc hin sao cho chy mu v tnh trng m bnh hc, v tr v thng tng t

nh trn ngi. Sau , nh gi tc dng ca thuc thng qua hai thng s chnh l

thi gian chy mu v khi lng mu chy. Thuc nu rt ngn thi gian chy

mu c coi l c tim nng trong cm mu khi c tn thng.

22

Tuy cc m hnh ch cho mc chy mu, v tr chy mu khc nhau, cn

v cc thng s o lng kt qu l ging nhau, nhng nhiu nghin cu th tc

dng ca thuc ln nhiu m hnh gy tn thng, thm ch c thuc ABS th

ln c 5 m hnh gy tn thng ny.

3.1.1. M hnh ct ui hoc chi.

Nguyn tc:

Mt thng s chn on khuyt tt ca h thng cm mu v nh hng ca

cc loi thuc n hin tng cm mu l khong thi gian m mu bt u chy

n khi ngng chy t mt vt rch tiu chun, c gi l thi gian chy mu. M

hnh ct ui hoc ct ct chi gy tn thng c ng mch v tnh mch l thch

hp nht nh gi tc dng ca thuc ln ton b qu trnh cm mu thng qua

thng s ny. M hnh ct ct ui c a ra bi Dttl v Ripke (1936) thng

c s dng ph bin nht trong thc nghim [91].

Cch tin hnh:

ng vt: chut nht trng Wistar c trng lng 250 450 g [19], [91] hoc

chut c Sprague-Dawley, trng lng 250 275 g. Ch c hai ging c s

dng. Cc con vt c chia ngu nhin thnh cc l:

+ L chng bnh: cho dng nc mui sinh l v ct ui hoc chi.

+ L th: cho dng thuc th nghim v ct ui hoc chi.

+ L so snh: cho dng thuc cm mu r c ch v ct ui hoc chi.

Gy m cc con vt. Kt thc thi gian hp thu thuc, tin hnh gy tn

thng bng cc phng php nh sau:

Gy tn thng chy mu theo phng php ct ui: t chut vo dng c

c nh chut, c l cho ui chut ra ngoi. Chut c cho dng thuc. Sau

mt thi gian xc nh, chut c ct ui vi mt li dao khong cch 2 -

4mm t chp ui. Sau khi ct ui, ngay lp tc ui c nhng vo mt cc

cha y dung dch mui ng trng 37oC [7], [69], [82], [91]. Subramaniam

(1996), Sogut (2013), Liu Y (2012), Rajasekaran (2010), White (2011) thc hin

phng php ct ui v tr 10 mm t chp ui [51], [93].

23

Gy tn thng bng phng php ct ct chi:

+ Trn chut: gy m chut bng ketamin 80 mg/kg. C hai chi sau ca chut

c cho vo mt cc cha 10 ml nc mui m ( 37oC). a chn ra khi nc.

Ct ct 2 chi sau 5 mm pha trn u gi [19].

+ Trn ch: co sch lng cc vng xung quanh mng chi. Bi m cho cc

vng bn di mng chn trnh mu chy vo cc v tr di mng chi. t chi

ch ln trn cc cnh ca bn m mu c ri t do. Mt dao ct c li trt

(Resco, Detroit, Michigan) c dng ct ngn chi ti v tr kt thc ca mng

(nh hnh v) [29], [66].

Hnh 3.1. V tr ct trong m hnh ct ct chi [29].

nh gi:

Thi gian chy mu: thi gian t khi ct ui/ ct ct chi cho n khi dng

chy mu (i vi m hnh ct ui quan st ti a trong 600 giy (10 pht)) [91],

Liu Y v cng s theo di trong 20 pht k c khi mu ngng chy, nu chy

mu ti pht sau mt khong dng chy mu, thi gian chy mu c xc nh l

tng thi gian chy mu trong 20 pht theo di [51].

Thi gian ng mu: m thng cu trc sau hc mt, thu thp mu vo ng

mao dn, bm ng h ngay lp tc cho n khi quan st thy mu hnh thnh cu

trc dng si th ngng [69].

Khi lng mu chy: ly lng mu thu c trong mi phng php gy

tn thng, tin hnh ly tm, thu hng cu. Ly gii hng cu, o quang hemoglobin

bc sng 550 nm [51], hoc tin hnh theo phng php cn bng trc v sau

khi gy tn thng nh m hnh gy tn thng tng (mc 2.1.1) [80].

24

3.1.2. M hnh gy tn thng da, nim mc

Nguyn tc:

Da trn nguyn tc nh m hnh gy chy mu cc mch mu. S dng mt

vt rch tiu chun bng cc thit b m bo s chnh xc v chiu di v chiu

rng ca vt rch vo nim mc ca con vt s pht hin c bt thng ca hin

tng ng mu hoc tc dng ca mt thuc ln qu trnh ng mu [58].

Klement v cng s (1998) m t mt m hnh chy mu tai th c

gy m, kim tra lng mu mt bng cch ly gii hng cu v nh lng

hemoglobin [41]. Forsythe v Willis (1989) m t phng php s dng m hnh

to vt rch trn nim mc ch nghin cu cc thuc cm mu trn c s phn

tch thi gian chy mu nim mc.

Cch tin hnh

ng vt: s dng ch cc tui khc nhau, c 2 ging [58], hoc th

trng New Zealand c hai ging, trng lng khong 2.9 3.8 kg [39], [41]. Chia

ngu nhin cc con vt thnh cc l:

+ L chng: dng nc mui sinh l v gy tn thng.

+ L th: dng thuc th nghim v gy tn thng.

+ L so snh: dng thuc cm mu r c ch v gy tn thng.

M hnh ny c th s dng nghin cu tc dng ton thn hay ti ch ca

thuc. Trong trng hp th tc dng ton thn, cho con vt ung/ tim thuc trc

khi gy tn thng. Trng hp th tc dng ti ch, dng thuc ln v tr rch.

Phng php gy tn thng:

Gy tn thng nim mc mi th: sau khi gy m, s dng mt li dao

sc, rch ng rch di khong 1 cm trn nim mc ca vch ngn trong mi ca

th [39].

Gy tn thng nim mc li ch: t con vt nm bn hoc v tr xng

c, mi trn c ln ra ngoi. Gn ming gc xung quanh c hm di v hm

trn mm. Dng mt li dao rch mt vt rch tiu chun vung gc vi mi,

ngay pha trn rng hm trn, hoc s dng thit b Simplate (c Forsythe v

25

Willis miu t nm 1989): t thit b ln nim mc, song song vi l mi, kch hot

thit b [58], [91].

Gy tn thng trn tai th: co lng vng tai v nhng tai trong mt cc

thy tinh c cha 1 lt dung dch nc mui, duy tr 37oC v lun c khuy duy

tr nhit . a tai ra khi nc v dng li dao Bard-Parker s 11, rch 5 vt

rch su ln tai (qua tt c cc lp ca da). Ngay lp tc cho tai vo cc ng nc

mui [41].

Gy tn thng trn da: i vi chut, gy m, co phn lng di chn.

Thc hin mt vt rch tiu chun (di 5mm, su 1mm) bng mt thit b t ng.

Mi 15s, p 1 giy lc vo vt thng trong 5s, cho n khi mu khng cn chy

na. Thi gian chy mu c xc nh sau 15s p ming giy lc cui cng [68].

i vi th: gy m, to 4 vt rch ln vng da lng th, mi vt di 10 cm, tin

hnh cm mu ti ch [97].

Gy tn thng m mm: to vt thng trn i chut cng trng bng cch

dng dao m ct, rch c i chut cng mt vt thng su 0.5 cm, di 1.5 cm [8].

nh gi kt qu

Thi gian chy mu: c hai cch hay dng: hoc s dng ng h bm gi,

bm thi gian t khi rch n khi vt rch chm dt chy mu [91], hoc thm mu

vo mt vng giy lc t di vt rch 1-2 mm cho n khi mu ngng chy, thay

i v tr ca giy lc mi 15s. Thi im c xc nh l kt thc chy mu khi

m giy lc khng cn thm vt mu ca mu [58]. Thi gian bnh thng ca

chy mu nim mc li ch t 2 4 pht [91].

Khi lng mu chy: sau thi gian 5, 10, 20 v 30 pht t khi gy tn

thng, mi thi im ly 10 ml dung dch nc mui (cha mu). Tch hng cu

bng cch ly tm 2000g trong 10 pht 23oC, b phn dch bn trn. Cho nc

mui vo phn t bo mu ny to hn dch th tch 1 ml, cho 10 l zapaglobin vo

ly gii cc t bo, chuyn hemoglobin thnh cyanmethemoglobin. o quang cc

mu ti bc sng 540 nh lng hemoglobin. So snh cc gi tr khi lng

mu mt ti mi thi im gia cc l [41].

26

Kim tra m bnh hc: mt mu sinh thit khong 0.5 cm, cha c ch rch

c ly sau 10 pht k t khi chm dt chy mu. C nh cc mu trong dung

dch formaldehyd 10%. Ct 2 mu vi dy mi mu l 5mm. Nhum v kim tra

cc mu di knh hin vi nh sng. nh gi v: fibrin trn vt rch v hng cu

thot mch. So snh hnh nh gia l th v l chng [39], [97].

So snh gi tr thu c gia cc l th nghin cu, phn tch thng k da

trn Students t-test.

3.1.3. M hnh gy tn thng cc tng

Nguyn tc:

Cc tng nh gan, lch, thn l nhng c quan ln bng v rt d b tn

thng [76]. Mt khc, chng c cu trc b mt rng, nhiu mch mu nui c

quan, nhu m khng c c trn, c t collagen, v vy khi b tn thng thng gy

chy mu rt nghim trng, rt kh cm mu [14], [43], [56], [70]. Chnh v gy

chy mu mc nng, cc m hnh gy tn thng cc tng thng s dng

th tc dng cm mu ti ch ca thuc. Tim nng cm mu ca cc thuc c

nh gi da vo cc ch s l thi gian chy mu v lng mu chy. ng thi,

kim tra m bnh hc bit c nh hng ca thuc ln vng m b tn thng.

Cc m hnh ny bt chc c cc tn thng hay phu thut tng thng gp

trn lm sng, c ng dng rng ri trong nghin cu [50].

Cch tin hnh:

S dng chut cng Wistar c trng lng khong 250 400g, hoc th

trng New Zealand c hai ging, nng 2.5 3kg, chia ngu nhin thnh cc l:

+ L chng trng: m bng m khng gy tn thng tng, khng c s

dng bin php cm mu no.

+ L chng bnh: gy tn thng tng v s dng dung mi pha thuc hoc

nc mui sinh l.

+ L th: gy tn thng tng v s dng thuc nghin cu.

+ L so snh: gy tn thng tng v s dng thuc cm mu bit r c ch.

27

M hnh ny thng gy chy mu mc nng, do , dng th nghim tc

dng cm mu ti ch ca thuc.

Sau khi chia l, cc con vt c i qua m v tin hnh gy m (chut:

tim mng bng ketamin hydroclorid liu 20 mg/kg v xylazin liu 2 mg/kg, th:

tim tnh mch natri pentobarbital liu 50 mg/kg) v c nh chng vo bn phu

thut. Theo di cc ch s m bo s khc bit l khng ng k gia cc nhm

v cn nng, khi lng phn tng ct b, cc thng s huyt hc nh (s lng

hng cu, hematocrit, hemoglobin, bch cu, tiu cu, aPTT, PT, TT) trc th

nghim v m bo khng c thay i v huyt hc no nh hng n thi gian

chy mu. Kim tra li sau th nghim nhm nh gi nh hng ca thuc ln cc

thng s trn. Ring trn th, t thm mt ng thng vo ng mch i theo

di huyt p ng mch trong sut thi gian th nghim, m bo khng c s khc

nhau gia cc nhm trc khi gy tn thng v s thay i huyt p do mt mu

sau tn thng.

Phng php gy tn thng:

Gy tn thng gan:

+ Trn chut theo phng php ct gan: Co lng phn gia bng, st trng

bng povidon iod. M khoang bng bng mt ng rch gia bng 3 cm theo

chiu dc, bt u t mi c. Bc l thy trung gian tri. Ct gan theo ng

ngang di nht trn thy trung gian tri m khng lm thay i v tr gii phu ca

thy. Khi lng gan ct vo khong 1 2g. Khu bng. Ring nhm chng

trng, ch kim tra gan sau bng c khu li [14].

Hnh 3.2. V tr ca ng rch trn m hnh gy tn thng gan [14]

Thy trung gian tri

ng ct

28

+ Trn chut theo phng php m thng gan: m hnh ca Murakamiet v

cng s: tip xc gan sau khi m bng, t giy lc cn trc trng lng di

thy gan. Gy mt vt thng trn gan (gia thy tri) bng kim tim 13 gauge

(hoc 18 gauge theo m hnh ca Tang v cng s (2010)) nh hnh di: [88],

[96]. Ngoi ra Akarsu v cng s (2011) cng thc hin m hnh gy vt rch (1cm

chiu di, 2mm chiu su) [12].

Hnh 3.3. Gy tn thng gan theo phng php m thng [96].

+ Trn th: thc hin 1 vt rch gia bng (di khong 20 cm). To tn thng

gan gy chy mu bng cch dng ko ct cc cc trc ca thy phi, tri v

thy gia ca gan. Cc tn thng gan ca cc con vt c to ra mt cch nht

qun.

Gy tn thng lch:

Sau khi lm sch vng bng ca chut bng dung dch povidon iod, m

bng bng ng rch di 3 cm. t mt bao polyester hnh phu di l lch

thu mu chy ra. To tn thng lch bng ng rch khong 0.5 cm chiu su v

0.3 cm chiu di [89].

Gy tn thng thn:

t chut v tr nghing, co lng vng lng, chun b dung dch povidon

iod st trng. Ct 1/10 qu thn phn y bng cch s dng mt bn mu

tiu chun ha t trc [43].

nh gi kt qu

Ngay sau khi gy cc tn thng nh trn, ty theo tng yu cu ca nghin cu m

c th nh gi kt qu qua cc thng s sau:

29

Thi gian chy mu: l thi gian t khi bt u thc hin vt ct ln b mt

cc tng cho n khi chy mu dng li hon ton. Duz v cng s (2010) cng a

ra bng gi tr thi gian chy mu cho cc v tr tn thng tng ng [23].

Th tch mu mt: Mu chy trong bng c thm vo bng cn trc

(sau khi mu chy hon ton, cho bng vo bng thm mu). Tnh khi lng

mu chy do tn thng bng cch gp bng thm mu v cn chnh xc. Mt

ca mu c gi nh l 1.0 g/cm3. Th tch mu mt xc nh theo chnh lch

bng ban u v sau khi thm mu, chia cho khi lng ng vt [50].

Cng thc: V = (mb2 mb1)/m

mb1: khi lng bng trc khi phu thut

mb2: khi lng bng sau khi phu thut

m: khi lng con vt

Tnh th tch mu mt trong tn thng lch: dng xi-lanh ly mu t ti

polyester. Tnh ton lng mu mt theo th tch trn xilanh v g ti polyester

khi khoang bng. Khu bng.

Xc nh cc cht ch im trong tn thng tng: ly tm mu mu thu

c huyt thanh, bo qun -80oC xc nh nng aminotransterase (AST),

alanine aminotransferase (ALT), lactate dehydrogenase (LDH), lactate v creatinin.

Cc nng c o theo cc my mc v dng c o c sn trong cc phng th

nghim [37], [76].

Cc xt nghim in-vitro: thi gian ng mu (CT), thi gian hnh thnh cc

mu ng (CFT), cng ti a cc mu ng (MCF) [77]. o aPTT v PT bng

my phn tch ng mu [50].

Kim tra i th: kim tra mc bm dnh trong bng, sau phu thut 7

ngy, gy m chut, m bng v quan st mc bm dnh ca vt thng vo

bng, bm dnh ca tng vi c quan xung quanh. Dnh: 1 im, khng dnh: 0

im. Tnh ton v cho im.

30

Kim tra vi th m bnh hc: git chut, ly cc mu m bnh hc v ngm

trong dung dch formalin 10%. Lm tiu bn. nh gi cc tiu ch m bnh hc

di knh hin vi (v hoi t, vim) v cho im theo bng sau:

Bng 3.1. im cc tiu ch hoi t v vim trn m bnh hc cc tng [14].

im Hoi t Vim

0 Khng hoi t 0% Khng vim 0%

1 Hoi t ti thiu 1% Vim ti thiu 1%

2 Nh < 25% Nh < 25%

3 Trung bnh 25% - 50% Trung bnh 25% - 50%

4 Nng > 50% Nng > 50%

3.1.4. M hnh gy chn thng no

3.1.4.1. M hnh to tn thng gy xut huyt no

Nguyn tc:

Chy mu no xy ra khi c tn thng cc thnh mch mu no, v khi

, vic cm mu l cc k cn thit nhng rt kh t tc dng cm mu.

Cc m hnh gy ra tn thng no bng cch to vt rch vo nhu m no c

dng nghin cu v chy mu no trn lm sng.

Mt khc, xut huyt v t mu no c th c o lng bng MRI, v

cc tnh trng vim, ph, xut huyt v thoi ha thn kinh so snh qua hnh nh m

bnh hc [24]. Chut c gy tn thng s no theo m t ca Stahel v cng s

(2000), ca Habgood v cng s (2007) l nhng m hnh kh thi trong cc nghin

cu cc thuc cm mu.

Cch tin hnh:

ng vt: s dng th New Zealand c trng lng khong 2.5 3 kg. Chia

ngu nhin thnh cc l:

+ L chng: gy tn thng no v cm mu ti ch bng dung mi pha thuc

hoc dung dch nc mui ng trng.

+ L th: gy tn thng no v cm mu ti ch bng thuc nghin cu.

31

+ L so snh: gy tn thng no v cm mu ti ch bng thuc cm mu

bit r c ch.

Phng php gy tn thng:

Gy tn thng no th: t cc con th v tr nm nghing, rch trn da

ng rch di 3 cm gia ng ni s-ui. Sau khi ct b mng xng, to 2 l

trn ng khp vnh (l mt ng khp ca vm s) di knh hin vi phu

thut. Khc nh nhng ln mng cng, gy tn thng vo su nhu m vi 4 mm

chiu di v 1 mm chiu rng [24].

Gy chn thng no chut: rch vt rch trn hp s, khoan bng mt mi

khoan phu thut to mt l khoan ng knh 4 mm, 1 mm pha bn v 1 mm

pha sau th bregma l ra mng cng. Cho mt thanh kim loi nng 250 g ri

xung mng cng t cao 2 cm pha trn. ng vt rch li. Tim di da dung

dch buprenorphine gim au. Cung cp thc n v nc ung t do [52].

Sau khi cm mu t yu cu, cc cnh ca mng cng c khp li m

khng khu. ng da pha trn li.

nh gi kt qu:

nh lng th tch mu t trong no sau 24h: s dng chp cng hng t T2

(MRI T2) l mt cch o th tch mu t ni s [48].

nh gi mc ph: Chp MRI trn mt phng ng khp vnh. nh

gi da vo gi tr T1W v T2W ca MRI. Mc ph c nh gi thng qua s

gim tn hiu trn T1W v tng tn hiu trn T2W. Din tch ca khu vc ph c

o qua hnh nh MRI.

Kim tra m bnh hc: 24 gi sau tn thng, cc con vt c git bng

tim tnh mch pentobarbital 100 mg/kg. Tch no nh gi m bnh hc. nh

gi: xut huyt, ph n quanh t bo, vim, v thoi ha thn kinh. Mi thng s

trn c phn loi theo cc cp 0: khng; 1: nh; 2: c ngha. Tnh tng s

im trong mi nhm v im trung bnh cng. So snh gia cc nhm.

32

3.1.4.2. M hnh gy xut huyt ni s (ICH) bng collagenase

Nguyn tc:

Khi b xut huyt ni s, nng enzym collagenase ti ch tng do c

gii phng t cc t bo v cc m sau khi b tn thng. Tc dng ca enzym

collagenase l ph v thnh mch mu, lm mu r r vo cc m xung quanh. T

nhng hiu bit ny, cc m hnh gy xut huyt ni s bt chc ICH t pht bng

cch s dng enzym collagenase tim ti ch vi liu thch hp [53], [55].

Cc phng php ny c nhiu nh nghin cu s dng. Illanes v

cng s (2010) ch ra rng chp cng hng t (MRI) l mt tr s o lng cho

khi lng mu t ni s, cng nh o lng mc ph no [36]. Foerch v

cng s (2013), Lauer v cng s (2013) [48], Sun v cng s (2011) [84] cng gy

ICH trn chut, tuy nhin, phng php o khi lng mu xut huyt bng cch

trc tip hn: nh lng hemolobin trong mu xut huyt no, o kch thc mu

t thng qua ct lt no v chp nh [8], [26].

Tin hnh:

ng vt: s dng chut c trng thnh C57BL/6 hoc CD-1, c trng

lng khong 20 - 27g hoc chut Wistar, c hai ging, c trng lng trung bnh

240 30g. Gy m bng halothan ng h hp (1.5% n 2%) trong hn hp

oxy/khng kh [26], [36].

Cc con chut c chia thnh cc l:

+ L chng: gy ICH v cho dng dung mi pha thuc hoc dung dch nc

mui ng trng.

+ L th: gy ICH v cm mu ti ch bng thuc nghin cu.

+ L so snh: gy ICH v cm mu ti ch bng thuc cm mu bit r c

ch.

M hnh ny c th s dng nghin cu tc dng ti ch hay ton thn ca

thuc. Th nghim tc dng ton thn yu cu tim thuc trc khi gy chy mu,

th nghim ti ch gy chy mu trc, sau th thuc.

33

Phng php gy xut huyt ni s chut: t chut vo khung rn (Model

51650; Stoelting) c nh chut. Khoan mt l khoan vi su 3.5 mm v tr

0.5 mm pha trc v 2 mm pha bn so vi v tr th bregma. t mt kim tim 10

l vo th vn cn li su 3.5 mm tnh t hp s. Sau , tim 0.3 ml dung

dch nc mui c cha 0.045 0.075U collagenase type VII. Nm pht sau, rt

kim, bt kn cc l khoan bng sp xng v khu da u li. Phu thut ko di t

15 35 pht cho mi con chut [26], [36], [55].

nh gi kt qu:

24h sau khi gy tn thng, ty vo tng yu cu ca nghin cu m c th

xc nh cc thng s sau:

nh lng khi lng mu xut huyt ni s: s dng phng php quang

ph hemoglobin sau khi gy ICH. Khi lng mu xut huyt sau mt khong thi

gian xc nh k t khi gy ICH [26], [48].

Phng php nh sau: gy m su bng isofluran 5%. Bc l v tch no chut,

chia thnh bn cu no tri v phi, c t trong ng thy tinh c cha 3ml m

phosphat, thu ly mu t no. Tin hnh siu m ly gii mng hng cu v tch

ly phn dung dch bn trn, v cho vo thuc th Drabkins. Xc nh t l hp th

quang 540 nm, tnh khi lng xut huyt no (c 2 bn cu) da vo ng cong

tiu chun [26].

o kch thc t mu ca chut: bc l v tch no chut, ct lt 1mm. Chp

cc hnh nh ca cc lt ct vi mt my nh k thut s. Kch thc khi mu t

c tnh qua cc hnh nh bng phn mm ImageJ (http://rsb.info.nih.gov/ij/) [26].

nh lng th tch mu t trong no: phng php MRI nh trn.

o ph no: sau khi chp MRI, git chut. G no v tch thnh hai bn

cu no. Cn ring mi bn cu bng mt cn phn tch in t xc nh trng

lng t. Sau , c hai bn cu c sy kh 70oC trong 48h v cn mt ln

na xc nh trng lng kh. Tnh ring cc gi tr khi lng mu t t v

kh [36].

34

Tnh ton lng nc theo cng thc sau:

(WWc DWc)/ WWc 100%

+ WWc: trng lng t hiu chnh: trng lng t ca bn cu tr i trng

lng mu t t.

+ DWc: trng lng kh hiu chnh: trng lng kh ca bn cu tr i trng

lng mu t kh.

Lng mu xut huyt v kch thc khi mu t c so snh gia cc

nhm vi cc t-test.

3.1.5. M hnh gy tn thng cc mch mu

Nguyn tc:

Mu chy ra khi mch mu (ng mch, tnh mch, mao mch) khi c cc

vt thng trn thnh mch v ch ngng li khi c s hnh thnh thnh nt tiu cu

do kt tp tiu cu dn ti hnh thnh fibrin ti vt thng . Thuc c tc dng ln

qu trnh cm mu lm rt ngn thi gian mu chy trn cc mch mu u c

coi l c tim nng cm mu. Da trn nguyn tc ny c th to cc m hnh gy

tn thng cc mch mu kim tra tc dng cm mu ca cc thuc mi [91].

Cch tin hnh:

ng vt: s dng th c New Zealand trng, trng lng 2 kg [32], hoc

chut nht trng, ging c Wistar (WA) hoc Sprague Dawley, trng lng 250

300g [38], [91]. Chut c chia ngu nhin thnh cc l:

+ L chng: gy tn thng mch mu, dng nc mui sinh l.

+ L th: gy tn thng mch mu, dng thuc th nghim.

+ L so snh: gy tn thng mch mu, dng thuc cm mu bit r c

ch.

M hnh ny cng c th th thuc dng ton thn hoc ti ch. Th thuc

ton thn khi gy tn thng cc mch mu nh v th thuc ti ch khi gy tn

thng trn cc mch mu ln. Phng thc th thuc nh m hnh gy tn thng

gan.

35

Trc khi gy tn thng mch mu, tin hnh gy m cc con vt (th: tim

bp ketamin liu 50 mg v xylazin liu 20 mg v duy tr bng natri pentobarbital

liu 15 mg/kg thng qua tnh mch bin tai; chut: tim mng bng natri

pentobarbital liu 60 mg/kg).

Phng php gy tn thng:

Gy tn thng tnh mch ch di th: rch mt vt rch phn gia

bng di. C lp tnh mch ch di khi cc m xung quanh, t nht 3 cm ca

mch c t ln tm nilon sng mu quan st r v tr gy tn thng. Lu

lng mu ca tnh mch ch di tm thi c gi bi 2 kp hai u. Thc

hin mt vt rch dc di 7 mm trn thnh mch chn [32].

Gy tn thng tnh mch i: rch da, bc tch m. m vo phn gia tnh

mch i sau bng kim, to tn thng c ng knh 0.6 mm [63].

Hnh 3.4. V tr m kim v ng knh vt thng trn tnh mch i [63].

Gy tn thng tnh mch cnh th: c lp cc tnh mch cnh khi cc m

xung quanh. Dng cy kim 23 gauge m vung gc thnh mch. Mu chy ra t

vt thng c thu vo dng c t bn di tnh mch [17].

Gy tn thng ng mch ch bng trn chut:

Rch ng gia bng bng k thut v trng, bc l cc phn sau phc mc

v tip xc ng mch ch bng. To vt thng bng cc s dng mt mi dao

phu thut m vo ng mch ch bng, gn n v tr phn nhnh xng chu

[38].

Gy tn thng ng mch i chut: bc lc ng mch i. Ct ng

mch i bng dao m, tin hnh cm mu ti ch v nh gi [96].

36

Gy tn thng ng mch mc treo: t chut ln bn m 37oC. M bng,

nng mc treo rut c ln bc l cc ng mch mc treo. Mc treo c t

trn trn mt tm nha v cung cp lin tc dung dch Tyrode 37oC. Dng kim

tim 25 gauge chc mt l thng trn ng mch. Xc nh thi gian chy mu i

vi ng mch mc treo nh (ng knh ngoi 125 - 250m) ng ni ca mc

treo rut vi rut [91].

Trong trng hp khng cm mu c (mu vn chy sau khi tin hnh

dng thuc ti vt thng), cn phi khu vt thng t c yu cu cm mu.

Cc con vt c cho n v ung nc y kim tra hnh nh mch mu sau

mt thi gian.

nh gi kt qu:

Ty theo tng nghin cu, c th nh gi kt qu qua cc thng s sau:

Thi gian chy mu ca cc con vt trong mi nhm: thi gian chy mu

c o bng mt ng h bm gi, c nh ngha l thi gian t khi lm thng

mch mu n khi mu ngng chy [38]. i vi cc ng mch mc treo: quan st

chy mu qua knh hin vi mc khuych i 40x [91].

Khi lng mu chy: t giy lc di cc v tr tn thng thm mu

chy xung. Thay th giy lc mi 15 giy mt ln. Cn chnh xc giy lc trc v

sau phu thut tnh ton khi lng mu mt (nh m hnh chy mu tng) [96].

nh gi tnh trng hp mch v huyt khi trn th: mt thng sau khi dng

thuc, chp x-quang mu lu thng qua mt on tnh mch nht nh (tim cht

cn quang iodid thng qua cc tnh mch i phi). nh gi qua hnh nh x-quang

[32].

nh gi m bnh hc: git chut vo ngy th 7 ca nghin cu. Ct mu 1

mm ng mch ch bng quanh v tr vt thng nh gi. C nh mu trong

dung dch formaldehyd m phosphat 10% trong 24h nhit phng. X l m

bnh hc, nhum mu v kim tra di knh hin vi quang hc phng i 40x.

37

Cc tiu ch nh gi: mch mu (vim, hoi t trung gian, hnh thnh nt

fibrin, tp hp hng cu), quanh mch mu (vim, hemosiderin, m ht). So snh

hnh nh m bnh hc di knh hin vi ca l th v l chng.

3.2. M hnh gy ri lon thnh mch.

Giai on thnh mch l mt trong nhng giai on u tin v quan trng

trong qu trnh cm mu. Khi c tn thng chy mu, phn x u tin l co mch

hn ch mu chy t vt thng. Nhiu bnh l gy gin mch hoc gy vim

lm co mch km, thay i tnh thm thnh mch dn n mu chy khi thnh

mch, th hin r rt cc mao mch. Do , nh gi mt thuc cm mu theo

c ch ln tc dng thnh mch, c th s dng cc m hnh sau:

3.2.1. M hnh gy gin mch bng acetylcholin

Nguyn tc:

Khi thnh mch b tn thng, cc c trn mch mu co li lm ng knh

ca mch mu gim xung. Trong nhiu bnh l, mch mu b gin, khin cho

thnh mch co khng tc dng cm mu. Mt khc, tc dng co mch l khc

nhau cc mch c ng knh khc nhau: tc dng co mch xy ra trn cc mch

c ng knh nh l mnh v r rng hn trn cc mch c ng knh ln [10].

Da vo cc nguyn l trn, to m hnh gy gin mch bng acetylcholin v la

chn 5 mch c ng knh khc nhau, kim tra s thay i ng knh mch mu

sau khi dng thuc di knh hin vi quang hc, ng thi tnh ton lu lng mu

qua on mch . M hnh ny thch hp nh gi tc dng cm mu ca thuc

theo c ch co mch.

Cch tin hnh:

ng vt: s dng chut c Syria vng c hai ging, trng lng khong

60 85 g. Chia ngu nhin cc con chut thnh cc l:

+ L th: chut c cho dng thuc th nghim.

+ L so snh: chut c cho dng thuc co mch bit c ch (c th s

dng cc thuc nh: adrenalin, noradrenalin, vasopressin...)

Gi tr trc khi nghin cu mi l c coi nh l chng cho chnh n.

38

Gy gin mch bng thuc acetylcholine: theo Knight v cng s (1991),

liu acetylcholin 0.01 - 0.05 g/kg tim ti ch gy khng lm thay i nhp tim

ng k m ch gy gin mch. Liu cao hn gy ra phn ng co mch d di, sau

gin mch. Do vy, la chn liu 0.01 0.05 g/kg tim vo vng da trn lng

[42].

Tin hnh t mm knh trn vng da lng chut ( quan st di knh hin

vi): gy m chut, t chut vo mt dng c c nh, khng lm che khut phn

lng chut. Ci khung titan vo lp da lng. Rch cc ng trn da theo hnh

khung titan v lt lp da gia khung thy r cc mch mu (tch b lp da v

cc m c cho n khi ch cn mt lp c mng m qua , cc ng mch v tnh

mch nh c quan st r rng m khng b tn thng) . t phin knh thy tinh

che ph bn trn khung (i km khung titan, c ng knh 12 mm, nh hnh v).

Knh hin vi cho php quan st cc ng mch nh (ng knh 156 23m) v

cc tnh mch nh (365 64 m) khi chng nm song song vi nhau [28], [73].

Hnh 3.5. Vt knh trn da chut quan st cc mch mu di knh hin vi [28]

Phn loi cc tiu ng mch nh sau: [73]

A0: ng mch nh, ng knh 156 23 m

A1: Tiu ng mch ln, ng knh 59.5 11 m

A2: Tiu ng mch nh, ng knh 25.6 6.4 m

A3: Tiu ng mch ngang, ng knh 10.2 2.5 m

A4: Tiu ng mch u ra, ng knh 8.6 1.8 m

39

nh gi:

o ng knh mch (D) cho tng cc tiu ng mch A0, A1, A2, A3, A4:

t phn mu to ln knh hin vi c trang b n quang Halogen F0-150.

S dng b lc mu xanh 420 nm nng tng phn ca hnh nh. La chn

hnh nh r nt nht, o gi tr trc v sau khi dng thuc th nghim.

Tnh ton lu lng mu ng mch theo cng thc:

Q = V (D2 0.012/4)

Vi tc (V) c hiu chnh theo kch thc mch c vn tc trung bnh

ca hng cu.

Xc nh gim ng knh mch mu, lu lng mu ng mch tng

l (gi tr trc v sau khi dng thuc). So snh cc gi tr trn gia 2 nhm

3.2.2. M hnh gy tng tnh thm thnh mch bng hp cht 48/80

Nguyn tc:

Tnh thm thnh mch tng s lm mu t thnh mch i ra cc m xung

quanh. Gy tng tnh thm thnh mch bng cch tim hp cht 48/80, sau nhn

bit tc dng tng tnh thm thnh mch qua s thot mch ca dung dch xanh

Evan vo vng m xung quanh. Thuc c tc dng gim tnh thm s lm gim m

ca dung dch xanh Evan thot ra v s c tc dng cm mu trong giai on

thnh mch [91].

Cch tin hnh:

ng vt: s dng chut c Sprague-Dawley vi trng lng c th t 160

- 200g, chia ngu nhin thnh cc l:

+ L chng: dng dung mi pha thuc hoc nc mui sinh l.

+ L th: dng thuc nghin cu tc dng cm mu.

+ L so snh: dng thuc cm mu bit c ch gim tnh thm thnh mch.

Co lng vng bng chut, tim tnh mch dung dch xanh Evan 1% vi liu 5

mg/kg. Mt gi sau, cc con vt c cho dng thuc. Kt thc thi gian hp thu

(30 pht sau), gy m nhanh bng ether v tim trong da 0.05 ml dung dch hp

40

cht 48/80 nng 0.01% c 3 v tr phi, tri v trn bng. Sau 90 pht, git

chut bng ether ri tin hnh o ng knh vng da b nhum xanh hoc ct vng

da b nhum xanh em ngm vo dung dch natri clorid 0.9%, sau khong 24 gi,

lc ly dch ri em o quang.

nh gi:

ng knh vng m dung dch nhum Evan thm nhp: vng da bng c

tim xanh Evan c ly ra, o lng bng thc o mm. Tnh gi tr trung bnh

cho tng l.

Tnh nng dung dch xanh Evan thot mch.

Phn trm c ch tng tnh thm ca thuc th nghim trn chut tim hp

cht 48/80.

3.3. M hnh gy ri lon tiu cu

Tiu cu l yu t c vai tr rt quan trng trong c ch cm mu. Khi tiu

cu gim s lng hoc chc nng th gy chy mu, biu hin l thi gian chy

mu ko di v cc xt nghim ng mu bt thng. Da trn nguyn tc ny m

cc nghin cu a ra nhng m hnh gy gim tiu cu nh gi tc dng

ca cc thuc cm mu.

3.3.1. M hnh gim s lng tiu cu

Gim tiu cu c th do nhiu nguyn nhn: gim tng hp (suy ty xng)

hoc tng ph hy (chiu x ung th, khng th khng tiu cu). Trn thc nghim,

c th s dng s kt hp nhiu phng php gy gim s lng tiu cu v bt

chc c cc trng hp gim tiu cu trn lm sng nghin cu cc thuc

cm mu.

3.3.1.1. M hnh gy gim tiu cu bng khng huyt thanh v chiu x

Nguyn tc:

Gim tiu cu c th gy ra bi phng php chiu x v tim huyt thanh

khng tiu cu. Chiu x lm gim sn xut mu tiu cu v huyt thanh khng tiu

cu lm tng ph hy tiu cu, kt hp hai phng php ny gy gim tiu cu r

41

rt trn th (Blajchman (1979)). Da vo nguyn tc trn c th to ra cc m hnh

gim s lng tiu cu nhm nghin cu tc dng cm mu ca thuc [17].

Cch tin hnh:

ng vt: th trng New Zealand c hai ging, trng lng trong khong 2.5

3kg. Chia ngu nhin thnh cc l:

+ L chng trng: gy gim tiu cu, khng nhn thuc iu tr.

+ L chng bnh: gy gim tiu cu, cho dng dung mi pha thuc hoc

nc mui sinh l.

+ L th: gy gim tiu cu v cho dng thuc th nghim.

+ L so snh: gy gim tiu cu v cho dng thuc c tc dng lm tng s

lng tiu cu (romiplostim hoc eltrombopag).

Gy gim tiu cu:

Chiu x: gim tiu cu c to ra bng cch chiu ngun cesium 90 rads

trong 30 pht vo cc con th, thc hin trong 10 ngy lin tip. Trong qu trnh

chiu x, tai v c th c bo v bng mt di ruy bng trnh gy hi cho cc

tnh mch c v cc mch mu tai. S lng tiu cu bt u gim sau khi chiu

x 4 ngy v gim r rt sau 10 ngy chiu x. Do , qu trnh chiu x thng

c thc hin trong 10 ngy.

Tim huyt thanh khng tiu cu (anti-GPIb, anti-IIb3, v anti-hlL4R):

tim cho th vo ngy chiu x cui cng, liu lng nm trong khong 0.04 v 0.1

ml/kg [17]. Goerge v cng s (2008) dng liu 2 g/g [30].

nh gi:

Xc nh thi gian chy mu tnh mch cnh: sau 30 pht tim huyt thanh

khng tiu cu. (theo phng php m t mc 2.1.3).

Xc nh thi gian chy mu vi mch: rch vt rch tiu chun 6mm qua tai

th sau khi c lm m trong 5 pht 37oC. Sau khi rch, ngay lp tc nhng

tai vo dung dch nc mui sinh l. Ghi nhn thi gian chy mu tai th.

S lng tiu cu: xc nh bng my m.

42

Cc xt nghim ng mu v chc nng tiu cu: PT, aPTT, TT, o kt

tp tiu cu (k thut nh mc 2.2.2.2) [17].

So snh s khc nhau gia cc nhm nghin cu v thi gian chy mu tnh

mch cnh, thi gian chy mu vi mch, s lng tiu cu.

3.3.1.2. M hnh gy gim tiu cu bng cc cht hot ha tiu cu.

Nguyn tc:

Khi tiu cu c hot ha tham gia to huyt khi, th s lng tiu cu

trong mu gim [91]. Do , to m hnh dng nghin cu tc dng cm mu,

c th tim tnh mch cc cht hot ha tiu cu nh: collagen, ADP. Cc cht ny

c th lm gim tiu cu ti a trong vng vi pht v hiu qu gim s lng tiu

cu c tng cng bng cch tim thm epinephrin [91].

Ngoi ra, Vlkl v Dierichs (1986) cho rng khi tim tnh mch enzym

collagenase dn n phn gii protein, hnh thnh cc khong trng t bo ni m

v tip xc su hn vo lp su ca thnh mch. Thnh mch b tn thng lm cho

tiu cu c hot ha kt dnh vi thnh mch, to huyt khi, do gy gim

tiu cu trong mu (mc ti a 5 10 pht sau khi tim collagenase) [91].

Da trn nguyn tc , gy gim tiu cu trn chut v tin hnh nghin

cu v tc dng ca cc thuc trn qu trnh cm mu.

Tin hnh:

ng vt: S dng chut lang c (Pirbright White), trng lng 300-600g,

hoc chut nht trng (260 300g) hoc th Chinchilla c 2 ging c trng lng 2-

3kg. Cc con vt c chia ngu nhin thnh cc l:

+ L chng: gy gim tiu cu v cho dng dung mi pha thuc hoc nc

mui sinh l.

+ L th: gy gim tiu cu v cho dng thuc th nghim.

Cho cc con vt dng thuc. Kt thc thi gian hp thu tin hnh gy gim

tiu cu bng cch tim tnh mch ui mt trong cc cht/ hn hp cht sau:

+ 1.5 U/kg collagenase [92]

+ 100 l hn hp collagen (150 g/ml) kt hp vi epinephrin (300 M).

43

+ 100 l hn hp collagen (150 g/ml) kt hp vi ADP (170 mM) [95].

Vi th: tim chm vo tnh mch vnh tai, vi chut: tim vo tnh mch

ui.

nh gi:

S lng tiu cu: ly khong 50 100 l mu vo ng cha kali-EDTA.

Xc nh s lng tiu cu ca mu mu ti cc thi im: 1 v 2 pht (chut), ti

5, 10, 15 pht (th) sau khi tim cc cht gy gim s lng tiu cu.

Tnh t l tiu cu trong l chng v l th ti cc cc thi im ly mu so

vi thi im ban u [91].

Tnh phn trm c ch tiu cu cho mi l th - chng [91].

3.3.2. M hnh gim chc nng tiu cu

3.3.2.1. M hnh gy gim chc nng tiu cu bng thuc khng tiu cu

Nguyn tc:

Di tc ng ca thromboxan A2, ADP tiu cu c hot ha, cc GP

trn mng tiu cu gn tiu cu vi ni mc v vi cc tiu cu khc. Cc thuc

khng tiu cu lm c ch hot ng ca cc cht hot ha tiu cu lm gim chc

nng ca tiu cu trong cm mu. Aspirin v cc thuc NSAID lm gim tng hp

thromboxan A2, clopidogrel c ch gn ADP vo receptor ca n trn mng tiu

cu, cn tr hot ha receptor GPIIb/IIIa dn n gim kt tp tiu cu [5], [10].

Trn lm sng, xut huyt ni s xy ra vi mt phn ba bnh nhn s dng thuc

khng tiu cu (Foerch v cng s (2006), Stead v cng s (2010)).

Da vo cc hiu bit trn, c th to m hnh lm gim chc nng kt tp

ca tiu cu bng cch s dng mt hoc kt hp cc tc nhn khng tiu cu sau

, nh gi trn cc m hnh chy mu, c bit l m hnh ICH. Cc m hnh ny

ph hp vi bnh l chy mu do qu liu thuc khng tiu cu trn lm sng, c

s dng trong nghin cu tc dng cm mu ca cc thuc.

Tin hnh:

ng vt: chut nht trng c 2 ging, trng lng (210 270g). Chut c

chia ngu nhin thnh cc l:

44

+ L chng trng: khng c dng bt k thuc g.

+ L chng bnh: cho dng cc thuc khng tiu cu, sau , cho dng

dung mi pha thuc.

+ L th: cho dng cc thuc khng tiu cu, sau , cho dng thuc th

nghim.

+ L so snh: cho dng cc thuc khng tiu cu, sau , cho dng thuc

cm mu r c ch o ngc tc dng thuc khng tiu cu (nh

antidote oligonucleotide 1 (AO1) v cyclodextrin-containing

polycation (CDP)).

Gy gim chc nng tiu cu: cho ung acid acetylsalicylic vi liu 10 mg/

kg mi 24 gi trong 4 ngy [45].

Gim chc nng tiu cu trong m hnh gy xut huyt ni s (ICH): s

dng aspirin liu 60 mg/kg mi 24 gi, hoc clopidogrel liu 22.5 mg/kg mi 24

gi, hoc c hai thuc, thng qua ng ung trong 3 ngy. Vi nng aspirin 0.4

mg/ml; clopidogrel 0.15 mg/ml, cho dng trong 72h, Moni v cng s (2005)

nghin cu c rng tng nng thuc khng tiu cu cng khng th gim hot

ng ca tiu cu hn na [49].

Gy ICH bng tim collagenase: tin hnh theo phng php nu mc

2.1.2.

nh gi kt qu:

Sau khi gy gim tiu cu, ty thuc vo yu cu ca mi nghin cu m c th

nh gi cc thng s sau:

Thi gian mu chy v khi lng mu chy: tin hnh o thi gian chy

mu ui chut theo phng php nu mc 2.1.5 Lng mu chy c thu li

v o hemoglobin bng cch o hp th [45].

Tnh % khi lng mu chy l c iu tr so vi cc l khng c

iu tr.

nh gi kt tp tiu cu trn in vitro: s dng my o kt tp tiu cu t

ng, nguyn l l: khi cho cht kch hot tiu cu nh ADP, collagen vo huyt

45

tng th tiu cu s b kt tp khng hi phc, lm thay i gi tr mt quang

hoc in tr ca dng in. Mc kt tp tiu cu c phn nh qua mc

thay i mt quang hoc in tr. Cc kt qu u ra bao gm: kt tp ti a,

dc ca ng cong kt tp, thi gian xut hin kt tp, thi gian t khi xut

hin kt tp n khi t kt tp ti a. So snh gi tr ca nhm th v nhm chng

[11].

nh lng ATP gii phng t tiu cu: o bng pht quang sinh hc

(Ingerman - Wojenski v cng s (1983). Thm vo mi mu 100 l luciferin-

luciferase ngay sau khi kt thc th nghim tp hp. Luciferin l mt cht s pht ra

nh sng khi c ATP trong dung dch. Thay i nh sng pht x pht hin nng

nh nht l 2 nM ATP, do , trong mi mu c cho thm lng 2 nM ATP.

Lng ATP do thuc gy gii phng t tiu cu trong cc mu c tnh bng cch

so snh vi ng cong chun ca 2 nM ATP. Nng ATP c a ra vi n

v l nM. So snh nng ATP gia cc mu [25].

nh lng serotonin (5-HT): s dng phng php ELISA. Cc mu mu

sau khi c ly t cc chut th nghim c acyl ha vi acetic anhydrid trong

aceton. Cho cc mu vo a 96 ging. Thm khng huyt thanh th (IgG th) v

qua m 4oC. Mu c c 405 nm trn u c ELISA (Molecular Devices

Union City, CA, USA). S lng 5-HT c nh lng theo tiu chun ca nh

sn xut v phn tch bng phn mm Origin (Microcal software, Northampton,

MA) [78].

Ngoi ra, ty nghin cu m c th xc nh thm cc thng s: hot tnh

enzyme cyclooxygenase, nh lng thromboxan A2, o dnh tiu cu [83].

So snh s khc nhau gia cc nhm nghin cu.

Cc m hnh khc: Hasgul v cng s (2012) cng cho ung aspirin trong

CMC liu 400 mg/ kg, sau 16 gi nhn n, cho ung trong 2 tun, gy chy mu

tiu ha, th thuc cm mu [43].

46

3.3.2.2. M hnh gim cht lng tiu cu do t bin gen lm gim yu t von-

Willebrand.

Nguyn tc:

Yu t von-Willebrand l mt cht keo sinh hc gn tiu cu vi collagen

lp t bo ni mc, v cng l yu t vn chuyn FVIII trong huyt tng [21],

[68]. S thiu ht v s lng v chc nng ca vWF gy bnh von -Willebrand,

thiu ht vWF s ko theo s thiu ht ca yu t VIII, do vy, trong bnh von

Willebrand th vWF v yu t VIII u gim.

Gen vWF ln, c 52 exon, nm trn cnh ngn ca nhim sc th 12 v tr

13.3. t bin bng cch xa cc nucleotid trong gen vWF gy gim s lng hoc

hot tnh ca vWF. C hn 300 t bin gen vWF c tm thy c th gy

bnh von-Willebrand [21]. Trong , thng gy bnh trn ch v chut bng cc

m hnh sau:

Ch bnh von-Willebrand: ch Scotlan Terrier l mt m hnh bnh von-

Willebrand c bit n ln u tin vo nm 1978. t bin gy bnh c gy

ra bng cch xa n nucleotid trong exon 4 ca gen von-Willebrand dn n

khng pht hin c mRNA v protein ca VWF. Sau , cc con ch c nhn

ging to bnh von- Willebrand nghim trng (type 3) c thiu hai alen, hoc

nh (type 1) c thiu mt bn sao ca gen.

Chut bnh von-Willebrand: khng ging nh ngi, nng von-

Willebrand chut l rt khc nhau. Chng chut thun RIIIs/J c nghin cu

nm 1990 c mc VWF l ~50% mc bnh thng c xut lm m hnh

bnh von-Willebrand type 1 cho ngi. Ngi ta xc nh c cc locus Mvwf

trn nhim sc th 11 lin quan n nng thp ca VWF. Locus ny to nn tnh

a hnh trong gen Galgt, l mt gen glycosyltransferaza. Gen Galgt b t bin lm

bt thng t bo ni m, thay i glycosyl ha i vi VWF gy ra gim nng

ca VWF trn ging RIIIS/J [71]. Chut dng RIIIS/J ko di thi gian chy mu

(hn 15 pht) so vi nhng con chut C57BL/6J bnh thng (1.8 pht) v cc

dng khc ca chut. Nng yu t VIII hot ha v khng nguyn von-

47

Willebrand chim 1/3 1/2 mc bnh thng. Huyt tng chut RIIIS/J cha

y cc multimer ca yu t von Willebrand, mc d nng mi multimer u

thp hn so vi chut bnh thng [87].

Nhng con vt ny c lu gi ging trong phng th nghim thc hin

cc nghin cu nh gi tc dng ca thuc ln ng vt mc bnh von-Willebrand.

Tin hnh:

S dng chut dng RIIIS/J b bnh von-Willebrand, t 2 8 thng. Cc

con vt c chia ngu nhin vo cc l:

+ L chng: cho dng nc mui sinh l.

+ L th: cho dng thuc nghin cu.

+ L so snh: cho dng thuc bit r c ch l lm tng vWF (nh

interleukin 11).

Ly mu: gy m chut, ly mu chut t m ri tnh mch sau h mt vo

ng nghim cha sn cht chng ng. Tin hnh nh gi mt s ch tiu.

nh gi kt qu:

Xc nh thi gian chy mu v khi lng mu chy: thi gian chy mu

ui chut c tin hnh theo m t ca Novak v cng s (nh mc 2.2.5) [87],

v thi gian chy mu da chut (tin hnh theo m hnh mc 2.1.4).

Xc nh cng thc mu [21] v cc xt nghim ng mu (aPTT, PT) [87].

o mc kt tp tiu cu: nh m hnh c ch kt tp tiu cu bng thuc

khng tiu cu [53].

Nng khng nguyn vWF: ly tm mu trong 15 pht ti 2500g nhit

phng ly huyt tng. S dng xt nghim min dch lin kt enzym (ELISA)

xc nh nng vWF, o quang bc sng 490 nm, s dng mu chun l

huyt tng gp t 10 con chut bnh thng [21], [22].

Xc nh hot ca FVIII: chng ng cho huyt tng bng EDTA. Pha

long huyt tng vi t l 1:40. Cho huyt tng vo ng nghim 37oC, sau 5 pht,

khi ng qu trnh ng mu bng vic cho thm CaCl2. Theo di qu trnh ng

mu trc quan ( 37oC), ghi nhn thi gian cn thit ng mu. Hot ca

48

FVIII trong cc mu c xc nh bng phng php ng chun (trc tung l

thi gian ng mu, trc honh l hot yu t VIII). S dng mu th l huyt

tng c hot yu t VIII bit r (mc bnh thng) [2], [21].

So snh gi tr gia cc nhm nghin cu.

3.3.2.3. M hnh gim cc ht trong tiu cu.

Nguyn tc:

Nm 1988, Novak v cng s nghin cu c c mi tng quan gia

gim sc t v thiu ht cc ht ca tiu cu. Tm trong 26 t bin sc t c gim

cc ht ca tiu cu lm thi gian chy mu ko di. Trong s , c 2 con chut

c miu t k cng l Sandy v Cocoa.

Sandy c t bin lm gim m sc, xy ra trn dng DBA/2J. Kim tra

di truyn cho thy nguyn nhn l do t bin gen ln trn NST s 13 gn locus ca

gen cr v Xt. y l kiu t bin th 10 trong cc t bin sc t chut km cc

khuyt tt trong melasosom, lysosom v cc ht ca tiu cu [86].

Cocoa c t bin lm nht mu lng, xy ra trn dng C57BL/10J thun,

c to ra phng th nghim Jackson vo nm 1978. t bin gen Cocoa l t

bin gen ln nm cui tm ng ca nhim sc th s 3 gn locus Car-2 [62].

t bin lm chut Sandy v Cocoa gim sc t mt v lng ng thi ko

di thi gian chy mu do thiu cc ht trong tiu cu (gim serotonin tiu cu,

gim tn sut cc ht khi quan st di knh hin vi). Mc serotonin ca tiu cu

rt thp mc d hot ng ca ATP l bnh thng. Khi quan st di knh hin vi

in t, s lng cc ht ca tiu cu gim ng k [62], [86].

Chut t bin Sandy v Cocoa c tnh trng bnh l tng t hi chng

Hermansky-Pudlak trn ngi, chng cng l m hnh thch hp cho hi chng ny.

Novak (1984, 1988) [62], [62], Reddington (1987), Lages (1987), Witkop (1989),

Weiss (1979), u cho rng gy t bin gim cc ht ca tiu cu chut v

ngi l ng nht. Cc con chut ny c nui v gi ging trong phng th

nghim.

Tin hnh:

49

ng vt: s dng mt trong hai dng chut sau nghin cu tc dng ca

thuc ln tiu cu gim chc nng (gim cc ht trong tiu cu):

Chut Sandy (SDY), chng DBA/2J, c to ra trong phng th nghim

Jackson nm 1983, con ging c duy tr trong ngn hng ging ca phng

th nghim Jackson.

Chut C57BL/10J t 2 5 thng tui, c t bin trn gen coa, dng ng

hp t (coa/coa).

Cc con chut c chia ngu nhin thnh cc l:

+ L chng: cho s dng dung mi pha thuc hoc nc mui sinh l.

+ L th: cho s dng thuc nghin cu.

nh gi kt qu: [61], [62], [86]

Thi gian chy mu: xc nh thi gian chy mu ui theo m hnh ct ui

mc 2.1.5.

Xc nh s lng tiu cu: tiu cu c m sau khi pha long vi dung

dch amoni oxalat 1% theo phng php ca Brecher v Cronkite.

nh lng serotonin cha trong tiu cu: Tiu cu c cho vo 1 ml nc

ct v ly gii. Nng serotonin c xc nh theo phng php hunh quang

theo Crosti & Luchelli (1962) (s dng bc sng kch hot ti 295 nm v pht x

ti 540 nm, nh lng bng cch so snh hunh quang ca cht chun serotonin

creatine sulfat) [61].

o kt tp tiu cu: ly mu, s dng cc cht gy kt tp tiu cu l

collagen v ristocetin, o kt tp tiu cu ti 37oC, bng phng php o in tr s

dng my o kt tp tiu cu trong mu ton phn, model 500. o ATP c tit ra

t tiu cu bng cch kt hp vi luciferin luciferase, o quang v so snh vi dy

cc nng ATP chun.

m s lng ht trong tiu cu: tiu cu khng nhum mu, lm kh trn

li carbon. Kim tra bng knh hin vi in t Hitachi H-600 ti 50kV, chp nh

tiu cu phng i ln 12000 ln. m s lng ht cha trong tiu cu c kch

thc > 50 nm. Tnh gi tr trung bnh.

50

So snh gi tr gia cc nhm nghin cu.

3.4. M hnh gy ri lon cc yu t ng mu huyt tng

ng mu l mt qu trnh ton vn, yu cu s c mt v tham gia ca tt

c cc yu t ng mu, vy nn, thiu ht cc yu t ng mu lm mu khng

ng c, dn n chy mu ko di. Da trn nguyn tc ny, cc nghin cu gy

thiu ht mt hoc nhiu yu t ng mu nh gi tc dng ca thuc cm mu.

3.4.1. M hnh gim cc yu t ng mu huyt tng bng heparin.

Nguyn tc:

Heparin c tc dng chng ng mu nhanh c in vitro v in vivo. Bnh

thng trong mu antithrombin III c tc dng chng ng mu do lm mt hiu

lc ca thrombin III v cc yu t ng mu IX, X, XI, XII hot ha. Khi c mt

heparin th n s to phc vi antithrombin III lm tng tc dng ca antithrombin

III ln 1000 ln, do , cc yu t ng mu v thrombin mt nhanh hiu lc lm

mu khng ng c [5], [10].

Heparin phn t lng thp (LMWHs) khng c ch thrombin m ch c ch

yu t X hot ha, do , trong cc xt nghim ng mu, LMWHs khng lm thay

i aPTT, nhiu, ch thay i hot tnh ca anti-Xa [10].

Da vo cc nguyn tc trn, cc nghin cu s dng heparin c dng

UFH v LMWHs to ra cc m hnh thiu ht cc yu t ng mu, s dng nghin

cu tc dng ca thuc.

Cch tin hnh:

ng vt: s dng chut c Sprague-Dawley c trng lng khong 250 -

274g. Chut c chia ngu nhin vo cc l: [47]

+ L chng bnh: gy gim yu t ng mu bng UFH/LMWH v cho dng

nc mui sinh l.

+ L th: gy gim yu t ng mu bng UFH/LMWH v cho dng thuc th

nghim.

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+ L so snh: gy gim yu t ng mu bng UFH/LMWH v cho dng thuc

cm mu bit c ch lm tng cc yu t ng mu trn (nh protamin,

yu t X).

Gy gim cc yu t ng mu bng heparin: tim tnh mch ui mt trong

hai cht gy gim yu t ng mu sau:

+ Heparin (UFH) liu 2.0 mg/kg duy nht

+ Enoxaparin liu 2.0 mg/kg duy nht.

Nm pht sau, cho dng thuc hoc nc mui sinh l, v tin hnh cc th

nghim sau: [47]

Gy chy mu bng ct ui chut: k thut nh mc 2.1.5.

Gy chy mu bng kp tnh mch: sau khi hon thnh th nghim chy mu

ui chut, bc l tnh mch ch bng bn phi. o lu lng mu bng mt u

d Doppler. Sau , kp cc tnh mch trong 1 pht v phc hi t nht 5 pht trc

khi tip tc kp, khi o lu lng bng Dopper cho thy cc tnh mch tc hon

ton th ngng.

nh gi:

Thi gian chy mu v khi lng mu chy: xc nh theo phng php ct

ui chut mc 2.1.5.

Thi gian tc tnh mch: thi gian t khi kp n khi tc tnh mch hon

ton.

Xt nghim ng mu: ly mu bng cch m vo tim, chng ng bng

dung dch natri citrat 3.8% ( 9 phn mu / 1 phn citrate). Ly tm ngay lp tc. Xc

nh cc ch s aPTT, TT [47], [67].

nh lng cc antiprotease (anti-Xa v anti-IIa): s dng Xa v thrombin

(IIa) ca con ngi t phng nghin cu v Amidolytic (Spectrozyme Xa v

Spectrozyme TH). nh gi nng anti-Xa v anti-IIa bng phng php o

quang [25], [67]. Vi cc con vt c gy bng LMWHs, ch cn nh lng anti-

Xa.

52

So snh gia cc nhm v s dng t-test xc nh ngha thng k ca

cc kt qu.

Cc m hnh khc: Sogut v cng s (2013) cng gy gim cc yu t ng

mu bng heparin liu 640 IU/ kg, tim mng bng 3 ln mt ngy, trong 3

ngy lin tip [80], Racanelli v Fareed (1992) dng heparin liu 1 mg/kg hoc

FraxiparinR liu 1 mg/kg [67].

3.4.2. M hnh gim cc yu t ng mu bng gy thiu vitamin K

Vitamin K c tc dng lm ng mu bng cch: carboxyl ha gc glutamat

trong cu trc ha hc ca cc yu t II, VII, IX, X chuyn chng thnh cc yu

t ng mu c hot tnh [2]. Gim Vitamin K s lm gim cc yu t ng mu

hot ha, gy chy mu ko di. Do , c th gy m hnh thiu vitamin K trn

thc nghim bng cch: cho dng thuc khng vitamin K, ch n thiu vitamin

K.

3.4.4.1. M hnh dng thuc khng vitamin K.

Nguyn tc:

Cc thuc khng vitamin K c ch enzym epoxid reductase, cn tr vic

kh vitamin K epoxid thnh vitamin K, do lm gim vitamin K trong huyt

tng. M hnh ny c Foerch v cng s thc hin nm 2008 trn chut

Wistar, s dng thuc khng vitamin K l warfarin, y c xem l m hnh hu

ch trong nghin cu tc dng ca cc thuc cm mu trn ng vt gim vitamin K

[26]. Illanes (2010), Schlunk (2012), Zhou (2011) cng s dng m hnh ny trong

nghin cu .

Cch tin hnh

Chut nht trng c hai ging, trng lng trong khong 200-350 g [19],

[27], [44], [48]. Cc con chut c chia lm cc l:

+ L chng trng: khng dng warfarin, sau cho dng nc mui sinh l.

+ L chng bnh: cho dng warfarin, sau cho dng nc mui sinh l.

+ L th: cho dng warfarin, sau cho dng thuc th nghim.

53

+ L so snh: cho dng warfarin, sau cho dng thuc cm mu r c ch

c th o ngc tc dng ca khng vitamin K (vitamin K, phc hp

prothrombin c c, huyt tng ti ng lnh, yu t VIII kch hot, axit

tranexamic) [36].

Gy chy mu bng thuc khng ng: chut c cho dng warfarin liu

nh sau:

+ 2 mg/kg cho 24h, pha vo nc ung, cho dng ko di 3 - 4 ngy [19], [26],

[27], [44], [48]. Kim tra gi tr INR ca cc con chut m bo p ng

vi warfarin l ng nht trn tt c cc loi ng vt trong nghin cu [19].

+ 10 mg/kg/ 24h, pha vo nc ung, mt hoc 2 ln trong ngy, trong 2 ngy

lin tip.

+ 0.4 mg/kg cho 24h, cho dng 3 ngy lin tip [36], [84].

+ 0.01 -1.0 mg/ 100g, tim mng bng trong 7 ngy [20].

i vi gy ICH bng cch tim collagenase: kim tra INR trc khi tim

collagenase, ch tim cho cc con chut c INR 3 6 [36]. Gy m hnh ICH bng

tim collagenase nh mc 2.1.2.

nh gi kt qu:

o INR: rt 0.6 ml mu t tnh mch. Cho mu vo ng nghim cha 66.6

ml citrat. o INR v PT bng my phn tch.

Thi gian chy mu: gy m bng isofluran 3%, duy tr nhit thn nhit

37 5oC. Thi gian c tnh t khi ct ct chn hoc ct ui hoc m thng

ng mch i tri bng kim 24 gauge n khi mu ngng chy, theo m hnh mc

2.2.5.

o thi gian mu ng: nh mt git mu (t vt thng chn hoc ui) ln

mt knh cong, dng ng h xc nh thi gian cho ti khi mu ng (khng thay

i hnh dng khi nghing phin knh) [8], [11].

Lng mu chy: o bng giy thm cho n khi mu ngng chy. Cn giy

trc v sau khi thc hin gy tn thng. Chnh lch trng lng giy trc v

sau khi gy tn thng c coi l lng mu chy [19].

54

M hnh gy ICH: nh gi cc kt qu nh nu mc 2.1.2.2.

3.4.4.2. M hnh gy bng ch n thiu vitamin K.

Nguyn tc:

Thiu viamin K trong mu xy ra trn nhng con chut c cho n mt ch

n c t hn 5 ng vitamin K/g mi ngy [57], [64]. y l m hnh c s

dng bi Matsuura v cng s (1988), Uchida v cng s (1987), Hirayama v cng

s (2007) v chng t y l m hnh hu ch cho cc nghin cu thuc cm mu

trn ng vt thiu vitamin K [57], [34].

Cch tin hnh:

ng vt: chut Dawley Sprague c, 7 tun tui. Nui trong phng vi

nhit 23 2oC, m tng i 55 10 %, chiu sng 12 gi mi ngy (8:00

20:00). Cc con chut c chia ngu nhin thnh cc l:

+ L chng trng: cho n ch n bnh thng v cho dng nc