I, Gii thiu qua v ebola v nghin cu

II, Tin hnh

1, Thit k v sang lc invitro cc siRNA
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Thit k cc siRNA c hiu m ch tc ng vo L,VP24, VP35, trong

:
SiRNA tc dng vo L th c m t trc
SiRNA tc dng vo VP24 v VP35 th c lm giu bng cch s
dng vectoer psiCheck 2

2, S gy nhin ebola vo tb vero e6

-

nh gi mRNA ca L, VP24, VP35 b ct bi RNAi th cho cc tb

Vero E6 nhim ebola sau 16h c cha tr vi cc SNALP(stabl nucleic
acid-lipid particle):
SNALP ch cha EK1-mod/VP24-1160 mod/VP35-855 mod
SNALP cha hn hp 3 loi trn
SNALP cha luc mod
Sau 24h tin hnh thu dch ni, tch chit v qRT-PCR.
Lp tb thu c cho dung gii vi trizol thu RNA tng s phc v
vic khuch i cDNA
SNALP: l nhng ht nh khong 120nm c s dng cung
cp siRNA tr liu cho c th ng vt c v. Trong SNALP th cc
siRNA c bao quanh bi 1 lp kp lipid

3, qRT-PCR
-

Tinh sch RNA bng kit

qRT-PCR nh gi mc hot ng ca gen

4, 5RACE (5 Rapid Amplification of cDNA ends)

-

s dung GSP (bng 1)

in di kim tra
kch thc d on ca cc sn phm ( hnh 3b)
EK-1 v VP24-1160 l 282 bp
VP35-855 l 205bp

5, Xt nghim kch thch min dch trong ng nghim

-

S dng cc t bo mu n nhn ca nhng ngi khe mnh hin

tng
Cc siRNA mod v unmod c th nghim kch thch min dch thng
qua nng IFN anpha( gii hn di 15.6pg/mL)

6, Diffirentiation (s bit ha) v tb CD34 hp th SNALP

Nui dng cc t bo gc sinh mu cung rn CD34 ca ngi trong 5

ngy
Cell media c thay c 3 ngy 1 ln
cc tb gc phn ha thnh CD11c, CD11b, CD14, b sung
interleukin 3,6 ti t hp ca ngi vo ngy th 6
n ngy th 10, cc t bo bit ha thnh hn hp dendrit cell v
macrophage
Cc dng tb nui cy c vs SNALP cha FITC(fluorescein
isothiocyanate)=> cc tb hp thu SNALP cha FITC s c nhn din
v tch ring

7, Nghin cu chut
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Chut ci (6-8 tun) CD1 ICR (n=16,4 con/ 1 nhm) nghin cu kch
thch min dch
Chut c(6-8 tun) v chut ci B6C3F1 (n=20, 5 con/ 1 nhm)nghin cu kh nng dung np
Nhng con ny c cch ly 1 tun trc nghin cu
SNALP vi 1 lng thch hp trong 0,2 ml PBS c tim ui vo cc
con chut:
Nghin cu kch thch min dch:
o Sau 4h tim, thu gan v huyt tng o nng IFN v
cytokine
o Gii hn di ca IFN hay interleukin 6 l 15,6 pg/mL
Nghin cu kh nng dung np: mu c phn tch ha hc lm
sng v hot ng ca alanine, asparate aminotransferase v
sobitol dehydrogenase

8, S ng gi lipid ca siRNA
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SiRNA c ng gp trong cc SNALP

Hn hp siRNA(cocktail) c ng gi cng nhau

9, Nghin cu trn linh trng khng phi ngi

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1 t hp cc siRNA bin i nhm ti gen ZEBOV trong SNALP: EK1mod, VP-24 1160 mod, VP-35 855 mod
Nhng con kh c cho nhim ZEBOV
Nghin cu th nht:
Anti-ZEBOV siRNA c a vo SNALP(2mg tng s siRNA/kg
mi liu) dng cho 3/ 4 con kh sau 30 pht k t khi chng
nhim ebola. Con i chng ko cho iu tr
3 con trn c b sung SNALP cha anti ZEBOV siRNA vi liu
nh c trong ngy 1,3,5.
Nghin cu th 2: ( iu tra thm cho chc chn mc d s tng cng
tn s phng php iu tr nghin cu 1 c th ci thin kt qu)

SNALP cha anti-ZEBOV siRNA(2mg/kg mi liu) c tiem cho 4/

5 con kh sau 30 pht nhim ebola. Con i chng c tim 1
liu tng t SNALP cha non-specific modified siRNA(luc mod)
30 pht sau nhim
Nhng con trn tip tc c tim nh vy trong ngy 1,2,3,4,5,6

III, Kt qu
-

Hnh 1:
Vp24-1160 l siRNA hiu qu nht trong vic lm tt cc gen
ZEBOV vp24
VP35-855 hiu qu nht trong vic lm tt gen VP35 ZEBOV
Vp-1160 ,VP35-855 cng EK-1 c s chn lm SNALP cocktail
Vic khng bin i ha hc lm gim hiu qu ca siRNA v th s
dng cc siRNA c bin i bng cch methyl ha chng. Nh vy
lm gim s kch thch min dch ca siRNA
Hnh 2:
Hnh a+b: SNALP cha luc SiRNA gy ra interleukin v IFN vi
nng cao trong khi PBS, luc mod ,cocktail th khng gy ra
kch thch min dch
Hnh c: SNALP cha PBS, luc mod, cocktail gy ra IFIT mRNA vi
nng thp, cn SNALP luc gy ra IFIT mRNA nng thp
Hnh D: (d qu ko vit vo y)
Hnh 3:
Hnh A: SNALP cha ZEBOV siRNA lm gim ng k sn phm
ca virut ebola trong nc ni thu t cc tb Vero E6 sau khi
nhim 48h
Hnh B: SNALP cha EK-1 mod/VP24-1160mod/VP35-855mod gy
ra mRNA c hiu c ct t mRNA ch ca t bo. Kch thc
3 sn phm pcr c d on chnh xc c mt trong t bo Vero
E6 c iu tr bng SNALP cocktail
ln 1 v 4 l PBS vs luc mod v th khng ln bng
2 ln cn li cha siRNA c hiu v hn hp siRNA v th
ln cc bng bng vi kch thc ng vi d on
Cc trnh t ca sn phm 5RACE pcr tng ng vi sn phm tch c
th ca mRNA bng cch xc nhn c c ch c bit ca RNAi
th hin rng anti-ZEBOV siRNA nhm ti nhng t bo nhim
ebola, th nh gi s hp th SNALP trong gan chut v trong t bo
mu CD34 ca ngi
Hnh 4: cc tb nhim ebola hp th mnh SNALP
Hnh 5:
Hnh A: alanine+aspartate aminotransferase v sorbitol
dehydrogenase l enzyme ca gan gan, o nng cc men gan
ny c th nh gi mc tn thng ca gan. Qua biu ta

thy, hot ng ca cc enzyme ny ko thay i sau khi tim

SNALP chng t kh nng dung np kh tt
Hnh B: s lng cc t bo mu cng khng b nh hng sau
khi tim SNALP