khang the don dong chong te bao mo va cau tra loi
TRANSCRIPT
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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
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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
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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/