Dissertation > Medicine, health > Internal Medicine > Infectious disease > Viral infections > Viral Hepatitis

Construction of Apoptosis-related Fas or TNFR1 Gene Interference Adenovirus Vectors in Fulminant Viral Hepatitis and Their Effects in Vitro

Author YeHuaLi
Tutor YangDaoFeng;NingQin
School Huazhong University of Science and Technology
Course Internal Medicine
Keywords fulminant viral hepatitis gene therapy miRNA Fas TNFR1 adenovirus vector
CLC R512.6
Type Master's thesis
Year 2011
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Objective: To investigate the interference effect of mFas or mTNFR1 in fulminant viral hepatitis (FVH), we construct pcDNA3.1-mFas expression plasmids and pcDNA6.2- mFas- mTNFR1-miRNA, and construct miRNA adenovirus expression vectors of mFas, mTNFR1 and mFas-mTNFR1.Method: We constructed two miRNA expression plasmids of pcDNA6.2-mFas- miRNA and pcDNA6.2-mTNFR1-miRNA. Then,miRNA Adenovirus expression vectors of mFas, mTNFR1 and mFas- mTNFR1 were constructed by Gateway Technology. After infecting 293A cells, adenovirus were packaged and high titered adenovirus expression vectors were obtained. We investigated the interference effects of pcDNA6.2- mFas- miRNA , pcDNA6.2-mTNFR1-miRNA both in vitro and in vivo in an murine hepatitis virus type 3 induced fulminant hepatitis model.The inhibitory effects of Ad-mFas-mTNFR1-miRNA expression vector on Fas and TNFR1 expression was measured at both mRNA expression level with realtime PCR and protein expression level with Western Blot.Results: pcDNA3.1-mFas expression plasmids, pcDNA6.2- mFas-mTNFR1- miRNA , Ad-mFas-miRNA, Ad-mTNFR1-miRNA, Ad-mFas-mTNFR1-miRNA adenovirus expression vectors were successfully constructed. In vitro the constructed interference plasmids (pcDNA6.2-mFas-miRNA、pcDNA6.2-mTNFR1-miRNA) specifically inhibited their target genes expression including mFas and mTNFR1 at both mRNA and protein levels compared with controls. In vivo, pcDNA6.2-mFas-miRNA, pcDNA6.2-mTNFR1-miRNA improved animal survival from 0% to 11.1% and 20 % respectively. In vitro, Ad-mFas-mTNFR1-miRNA significantly inhibited mFas and mTNFR1 expression at both mRNA and protein levels. Conclusions: We successfully constructed pcDNA3.1-mFas expression plasmids, pcDNA6.2-mFas-mTNFR1-miRNA expression plasmid, and Ad-mFas-miRNA, Ad-mTNFR1-miRNA, Ad-mFas-mTNFR1-miRNA adenovirus expression vectors. Prelimilary experiments demonstrated their specific inhibitory effects on target genes expression and interference of Fas or TNFR1 significantly improved the animal survival in MHV-3 induced FVH.Significance: Fulminant hepatic failure is a severe liver disease with a mortality as high as to 80%~90%. Our study targeting multi proimflammatory genes expression may shed light on new approches for disease control.

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