Khang the Don Dong Chong Te Bao Mo Va Cau Tra Loi

8/14/2019 Khang the Don Dong Chong Te Bao Mo Va Cau Tra Loi

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GVHD: PGS.TS. Trn Th Dn


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Nhm thc hin

Nguyn Th Thu GiangV Th Mng Huyn

Phan Th Hng Thy Phm An Nhininh Th Thy HngTrnh Th Kim LinTrn Vn TrngTrn Ph DuyH Vn Cng


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Ni dung chnhI. Mc tiu chnh ci thin cht lng tht

II. Khng th chng li t bo m

III. Sn xut khng th n dng chng t bo m heo

IV. Tnh c ca khng th 4G7 i vi t bo m in vitro

V. Tim khng th 4G7 cho heo con

VI. Tm tt


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I. Mc tiu chnh ci thin

cht lng tht Tht c gi tr dinh dng cao t yu cu v k thut: d bo qun, ng nht, trnh

tnh trng oxid ha.

Cm quan: mu, mi v v cu trc. Tht gm: m c, m m, m lin kt

Pht trin khi c Gim khi m

nh hng mi l nghin cu k thut dngkhng th tiu hy khi t bo m v tin t bo

m khi th cn non.

Kch thch tKhng sinh

Khng th


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II. Khng th chng li t bo m1. T bo m


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(From: http://health.howstuffworks.com/fat-cell.htm)


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Khng th l cc globulin (Immunoglobulin- Ig) c trong

huyt thanh ca ng vt c kh nng lin kt c hiuvi khng nguyn kch thch sinh ra n.

2. Khng th


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IgG IgA IgM IgE IgD

V tr chyu

MuNim nhy

Cc dch tit

Lympho B

Mu

Bch cu ikim

T bo mast

Lympho B

T l 70-75%

15-20% cckhng th

trong huyt

thanh

10%


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3. Khng th a dng chng t bo m3.1. Khng th a dng

L mt tp hp cc khng th c hiu vi ccepitop khc nhau trn mt khng nguyn cho

trc.

Cc khng th a dng, mi khng thlin kt vi mt epitope khc nhau


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Khng th hay t bo T c hiu khng nguyn khng kt hpvi ton b cu trc khng nguyn m ch nhn din nhng

phn nh nht nh c cu thnh trong khng nguyn . V

tr nhn din ny c gi l epitop.


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B th l tp hp cc protein huyt tng khi c

hot ha s tiu dit cc vi khun xm hi bng cch

c cc l thng trn vi khun.

to iu kin cho:

hin tng thc bo.

thanh lc cc phc hp min dch.

phng thch cc phn t ha hng ng.


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3.2. Khng th a dng chng t bo m

Khng th a dng c to ra chng li t bom ca chut, th, b, cu, heo, g.

Tc dng:

Chut gim s lng m d tr trong c th, tng

trng c ci thin.

Th cng gim khi m lng v m vng thn. Cugim lng m di da, lng v quanh thn. Tuy

nhin khng c s ci thin r rt khi lng quy tht.


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Vn :

In vitro, loi khng huyt thanh ny lm v hng cu

Cho phn ng cho vi m khc cng vi m m.

Lm xut hin phn ng chong phn v (phn ng st, rung,au bng) khi tim vo (gii quyt bng cch tim lp li

nhiu ln vi liu thp).

Khng th ti sn xut loi khng huyt thanh ny.

Khng th n dng l mt chin lc khc gii quyt vn

trn.


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4. Khng th n dng chng t bo m4.1. Khng th n dng

Cc khng th n dng ch nhn bit mt epitope trn mt

khng nguyn cho sn. Tt c cc khng th n dng cng

mt dng th ging ht nhau v c sn xut bi cng mtdng tng bo.

Khng th n dng, lin kt vi mtepitop c hiu


16/75Hnh2: Quy trnh sn xut khng th n dng


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4.2. Khng th n dng chng t bo m

Khng th n dng chut c to ra chng li

mng tng ca t bo to m.

Khng th ny khng cho phn ng cho vi bt k tbo m hay m khc.

S dng khng th n dng khng li t bo to m

kim sot s pht trin ca t bo m trn chut, g, th,gia scnh s c hiu cao ca khng th n dng

vi nhng protein b mt (khng nguyn) ca t bo m.


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Kh nng ca khng th n dng l gy c t bo thng

qua b th ln trn cht gian bo mch mu ca t bo m

heo nui cy in vitro, v gim s pht trin ca t bo to

m trn c th nhng heo con.

V d: Yu v ctv (1917) dng khng th AD-3 hy

tin t bo m thng qua phn ng c t bo trong mi

trng gian bo mch mu, cn nhng t bo khc c th

ti sinh bng mt x l c bit.


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III. Sn xut khng th n dngchng t bo m heo

B1:Chut c min dch vi t bo m

C 2 cch:

Quy trnh ngn ngy

Quy trnh di ngy


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Quy trnh ngn ngyKhng nguyn c tim 4 ln, mi

ln cch nhau 3 ngy chn sau

Ngy 12: ct ly hch bch huyt, thu

hoch bo tng.


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Quy trnh di ngy

Tim khng nguyn vo mng bng 2ln / thng / 4 - 6 thng.

Ct ly lch v thu hoch t bo lch.


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B2: Bo tng (hoc t bo lch) c hp

nht vi t bo ung th ty xng SP2/O trong

mi trng c polyethylene glycol v lp t

bo phc mc => t bo lai.

B3: To dng t bo lai trong mi trng

RPMI c 3% Hypoxanthine Aminopterin

Thymidine (HAT) v 10% huyt thanh thai b.


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B4: Xt nghim khng th trong dch ni bng

phng php ELISA.

Nu dch ni dng tnh vi t bo m => t bo n

dng trn c tip tc pht trin to lp t bo

lin tc trn ging nui cy.

Ti n dng t nht hai ln => to lp t bo lin tc

mi.

B5: Dch ni c xt nghim ELISA xem kh

nng phn ng i vi khng nguyn ca t bo m.


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Kt qu: Thu c 44 n dng p ng vi

tiu chun ny: 4 dng vn cn cho phn

ng dng tnh sau khi cho n dng v

ng lnh vi ln (1B5, 2C2, aG7, 3B12).

Phn ng ca khng th khng xy ra hoc

rt yu vi t bo khng c lipid hoc t bo

ni m t ng mch phi heo.


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Qua xt nghim nhn thy khng th dng

4G7 rt c hiu vi m m.

Tinh khit khng th 4G7 bng sc k ct i

lc vi protein A.

nh gi tinh khit bng in di SDS-

PAGE, nh gi hot tnh bng ELISA.


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Phng php in vivo: sn xut khng th nPhng php in vivo: sn xut khng th ndng bng phng php dch trn bngdng bng phng php dch trn bng

Chut c tim dng t bo lympho chuyn

bit vo trong c th chng.

Kch hot chut bng hai mi tim u c

cha pristane vo xoang bng, cch nhau 1

tun =>gia tng p ng sn xut khng th.

Pristane gy nn phn ng vim ht v tc

ng n qu trnh tch dch bng.


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Sau 2 tun tim vo xoang bng chut khong 106 t

bo lai => gy nn cc bu v kch thch tit dch

trn bng lm bng chut phnh to ln.

Thu dch trn bng bng phng php chc d sau

2 tun tim t bo lai.

Kt qu: thu c hm lng khng th trong dch trn bng rt cao, khong 2 -10mg khng th/ml dch

trn.


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IV. TNH C CA KHNG TH 4G7I VI T BO MIN VITRO

1. Xt nghim tnh c ca khng th n dng

Xt nghim khng th n dng trong mi

trng cha gian bo mch mu t m m.


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Cc th nghim

Wright v Hausman (1995)Cho thy khi c b th hin din th vi khng

th n dng nh AD-1, AD-2 gy c trn

t bo m heo trong mi trng nui cy c

huyt thanh.

Ngc li, khi dng cc khng th ny cng

vi nhau th s gy ra s cht t bo trong

mi trng nui cy t bo khng c huyt

thanh.


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Cc th nghim

Nm 1997, Yu et al s dng khng th n dng AD-3

xc nh tin t bo m. AD-3 lin kt vi khng

nguyn xut hin sm trong qu trnh hnh thnh t bo

m.


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Th nghim ca Remacle v ctv, mt mnh 4G7 gy c t bo m thng qua b th trong mi

trng cha insulin v hydrocortisone d c haykhng c huyt thanh.

Khng th 4G7 l dng IgG2b isotype.


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Cc m (m di da) v ri rc nhiu trong m

(trong c) c nh du bi immunoflourescencedng 4G7 nh khng th ban u trn b mt ng

lnh (Hnh 3).

Khng th khng tc ng ngay vo gan, thn, c

(Hnh 3), hay mch mu heo. Trong nui cy gian

bo mch mu ngun gc heo, nhng t bo ctm thy bi khng th l c cha 1 vi git m

(Hnh 4).


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Hnh3: Hnh nh hin vi ca phn ng lnh m cln, c nhum min dch vi khng th n dng4G7. Ch c nhng m m bn trong c mi b tiu dit.

Bar = 100 mm.(http://jas.fass.org)


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Hnh 4: Nui cy gian bo mch mu vi khng

th n dng 4G7 trong 7 ngy. Ch nhng tbo cha y cht bo mi lm bin mu mnhm (mi tn). Bar=100 mm. (http://jas.fass.org)


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V. CC NH HNG KHI TIMKHNG TH 4G7 CHO HEO CON

Tim khng th 4G7 vo phc mc heo Large White lc 2 v 5ngy tui heo c git m nhn thy:

Tim, lch, thn khng nh hng.

Khi lng ca cc m m u gim so vi khi lngquy tht.

M m di da v m lng gim ng k.

Kch thc t bo m di da nh hn.M trong c thn v tng trng khng nh hng.

Khng nh hng n tng trng v khi lng quy tht

khng thay i (Remacle v ctv, 2000).


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Hnh 5: T l lipid (%) so vi trng lng ca mti vi s kim sot x l trong 38 ngy.

(http://jas.fass.org)


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Hnh 6: ng knh t bo m (m) khi x lkhng th 0.1 v 1 mg/ kg khi lng c th th.

(http://jas.fass.org)


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Tm linh hng c ch ca khng th n dng

4G7 ln giai on chuyn ha ca t bo m lc 5tun tui th bn vng cho n khi th t trng

lng git m (khi lng c th khong 100kg).


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V. TM TT

Cc nh to ging pht trin chin lc gim lng chtbo trong tht bng phng php min dch chng s hnhthnh t bo m.Khng huyt thanh a dng

Khng th n dng c ch hn c, ch yu heo.

Remacle v ctv (B), nghin cu s dng khng th n

dng chut thuc lp ph IgG2b chng li mng tngca t bo m heo.

C th s dng khng th c hiu chng li t bo m lm gim khi m m t nh hng m c vn.


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TI LIU THAM KHO

1/ PGS.TS. Trn Th Dn. Cng ngh sinh hc trong

chn nui gia sc. Nh xut bn nng nghip 2005.2/ PGS.TS. Lm Th Thu Hng. Gio trnh gii

thiu min dch hc. i hc nng lm TP. H ChMinh.

3/ http://jas.fass.org
http://jas.fass.org/http://jas.fass.org/


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1. T bo m


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2. M m

L hnh thi thch nghi ca m lin kt tha lm

nhim v tch tr m, chng ta nhit v gim th

trng. M ny thng c nhng vng c nhiu mchmu: h b ca da, ty xngT bo m c th ri

rc hoc hp li thnh tiu thy, ngn cch nhau bng

m lin kt cha mch mu (stromal vascular).


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3. Tin t bo mCc nh nghin cu thuc Trung tm y t UT

Southwestern mi y pht hin ra rng t

bo m cha trng thnh, hay c th gi ltin t bo m, n nu trong thnh mch mu nui dng m m. Chng ch i ngun

calo d tha pht trin tr thnh t bo m.

S pht trin thnh t bo m


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S pht trin thnh t bo mT bo trung m hoc t bo

si

t bo m nguyn thu(adipoblasts)

Tin t bo mpreadipocytes

t bo m trng thnh(adipocytes)


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(http://www.imsb.ethz.ch/researchgroup/cwolfrum/resea

rch/Adipocyte_Differentiation)


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Cc nh nghin cu pht hin thy tin t bo

m bm trn thnh mc mu chy qua m m.

Nui cy nguyn thy nhng t bo gian bo

mch mu (stromal vascular cell) trong m m

ca heo ch yu cha tin t bo m

preadipocytes v nguyn bo si fibroblasts.
http://jas.fass.org/content/vol83/issue5/images/large/a4539G-4.jpeg


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Figure 4. Muscle stromal-vascular cells cultured on laminin, rinsedafter the first hour and treated with serum-free media with insulin,transferrin, and selenium (ITS) from d 3 to 6 following either fetal

bovine serum + dexamethasone (A) or pig serum (B) from d 0 to 3. Ond 6, cultures were either stained with a peroxisome proliferatoractivated receptor (PPAR ) antibody (A) or the AD-3 monoclonalantibody (B), a preadipocyte marker
http://jas.fass.org/content/vol83/issue5/images/large/a4539G-4.jpeg


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So snh phng php

in vitro v in vivo


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Phng php in vivo:

u im:

- Nng khng th cao.

- n gin, d lm.

Nhc im:

- Tn nhiu cng chm sc n chut

- C th ln protein chut hoc cc tp cht khc.


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Phng php in vitro:

u im:- Gim vic s dng chut, khng vi phm cc

chnh sch bo v ng vt.

- Thun tin cho vic sn xut ln.

- Gim nhn cng chm sc chut.

- Trnh c s tp nhim t c th chut.

- Nng sut cao


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Nhc im:

- Mt s t bo lai khng pht trin trong mi

trng nui cy

- S tp nhim xc t bo lai b cht, huyt

thanh thai b hoc cc khng th khc hindin trong mi trng nui cy.

- Gi thnh cao quy m sn xut nh v va.

- Yu cu cao v trang thit b v trnh

chuyn mn, kh trin khai rng ri.


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S dng phng php no?Nu s dng phng php in vivo: lng dch

trn bng thu c trn mi chut vo

khong 10 ml. Hm lng khng th thu c

rt cao: 2-10mg KT/ml dch trn.Tuy nhin Remacle v ctv c gng sn xut

4G7 in vivo nhng k thut ny khng hiu

qu.

Phng php in vitro: c th thu c 20mg

KT/ml.

Q h kh h d


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Qu trnh sn xut khng th n dng,dch ni cha ci g?Khi cn sn xut, t bo c r ng v nui

cy trn mi trng sn xut khng th ndng (mt RPMI 3% HAT + 10% huyt thanh

thai b).Trong qu trnh pht trin cc t bo lai s sn

sinh ra khng th v bi xut khng th ra bn

ngoi mi trng.

Xt nghim bng phng php no?


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Xt nghim bng phng php no?Xt nghim khng th bng k thut ELISA:

Khng nguyn c gn ln cc ging ca v thpolyvinyl chlorure.

Sau ging s c ph dung dch albumine

ngn cn s gn kt ca khng th vo cc im cnli trn ging (gip m bo KT ch gn kt c hiuvi KN tng ng c gn trc ).

Sau dch nui cy t bo lai c cho vo ging khong 60 pht KN-KT c th kt hp vi nhau.Phc hp KN-KT s c pht hin bng protein Ac nh du bng cht pht hunh quang hoc

enzyme to mu.


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Mi trng RPMI

RPMI-1640 MediumModified

Liquid

With sodium bicarbonate Without phenol red and L-glutamine;

supplement with 0.3 mg/L L-glutamine.

Sterile-filtered

Endotoxin tested

Cell culture tested

RPMI 1640 c pht trin bi Moore et. al. atRoswell ParkMemorial Institute

Phn bit vaccine, khng th n dng v


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Phn bit vaccine, khng th n dng vkhng huyt thanh

1. VaccineL ch phm sinh hc c iu ch t chnh tcnhn gy bnh (ton phn hay mt phn) hay

sn phm ca chng, khi c a vo c thi tng th khng c kh nng gy bnhcho i tng nhng u c kh nng kch

thch sinh min dch.Vaccine gm 2 thnh phn chnh: khng nguyn

cht b tr


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2. Khng th

Khng th l cc globulin (Immunoglobulin- Ig) c

trong huyt thanh ca ng vt c kh nng lin kt

c hiu vi khng nguyn kch thch sinh ra n.

Cc khng th n dng ch nhn bit mt epitope

trn mt khng nguyn cho sn. Tt c cc khng th

n dng cng mt dng th ging ht nhau v c

sn xut bi cng mt dng tng bo.


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3. Khng huyt thanh

Khng th c tm thy ch yu

trong huyt thanh ng vt. Huytthanh cha khng th c hiu khngnguyn gi l khng huyt thanh.

Phng php chc d:


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Phng php chc d:

Figure 1. Mouse showing swollen abdomen typical of ascites.

http://www.nal.usda.gov/awic/newsletters/v8n3/8n3mcard.html

Khng th n dng tiu dit hay c
http://www.nal.usda.gov/awic/newsletters/v8n3/8n3mfg1.htm


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Khng th n dng tiu dit hay cch t bo m?

Khng th n dng khi tip xc vi tin t bo m( khi m cha tch t trong t bo) khng th s :c ch s pht trin ca tin t bo m ngha l

lm cho tin t bo m khng bit ho thnh t bom c.tiu hu tin t bo m .

V d: Yu v ctv (1917) dng khng th AD-3 hy

tin t bo m thng qua phn ng c t bo trongmi trng gian bo mch mu (trch trong schCNSH trong chn nui )


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-Hnh 3a nui cy gian bo mch mu t m m khng

s dng khng th ( lipid c nhum bng du )-Hnh 3b nui cy gian bo mch mu t m m c xl khng th trong 24 gi th hu ht t bo m b tiuhu.Bar = 30 mm (http://jas.fass.org)

Phng php v liu lng khng lm


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Phng php v liu lng khng lmnh hng n lp m bng ca heo?

Theo cc nghin cu v khng th n dng chng tbo m th s nh hng ca KTDD l nhiu nht ti

nhng vng m c th hot ng nhiu nh i, bn,

vai, ngcnhng ni c m tch t nhiu.Cn lp m di bng, theo nghin cu cho thy

rng s tc ng ca khng th n dng v vng ny

t hn nhng vng khc, chng ch tc ng n s tt bo m y nn hu nh khng lm nh hng

n vai tr ca lp m ny i vi c th.


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Th nghim ca Wright v Hausman

Trong th nghim ca Wright v Hausman trong mitrng c huyt thanh v khng th n dng AD-1

hoc AD-2 v b th th t bo m heo b gy cNgc li, khi dng cc khng th ny cng vi nhau

th s gy ra s cht t bo trong mi trng nui cy

t bo khng c huyt thanh.

i vi th nghim ca Wright and Hausman


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i vi th nghim ca Wright and Hausmantrong mi trng khng c huyt thanh .S hin

din ca t nht hai loi khng th s dn qu trnhkch thch s cht ca t bo thng qua qu trnhtng tc vi nhng ha cht thm vo trong qutrnh nui cy.

Cn trong trng hp cn li (c huyt thanh cb th ) th trong mi trng nui cy h khngc thm corticosteroids dn n hin tngGlucocorticoids b gim st trong qu trnh nuicy>> s khuch i qu trnh qu mn t bo


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Phn bit cc loi khng thchng t bo m:AD-1; AD-2;

AD-3v 4G7


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Mt khng nguyn c th c nhn din bi

nhiu khng th vi c hiu khc nhau, dngkhng th no ph hp nht v cu trc 3 chiuvi epitope s c khuch trng mnh nht.


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Figure 6. Tissue lipids expressed as percentage of wetweight at 38 d in a control group and in groups treate

with .1 or 1 mg of antibody per kilogram of bodyweight. The data were analyzed with a three-way ANOVAollowed by Newmann-Keuls post hoc tests : *,P < .05; **,

( //j f )
http://jas.fass.org/http://jas.fass.org/

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