The Role Of Interferon In2’-5’OAS Promoter Regulated Recombinant Caspase-3Plasmid Inducing Hepatocyte Apoptosis

Background and Objective:According to the statistic of the World Health Organization, about3%people infected with hepatitis c virus (HCV) in the world,170million chronic carriers might have the possibility of developing into liver cirrhosis and hepatocellular carcinoma (HCC). Prevention and control of hepatitis becomes a major problem we confronted, and HCV makes its control more difficult because of its easy mutation. So far, there are no effective methods to produce anti HCV drugs and vaccines to prevent and control HCV infection, especially HCV RNA which is very difficult to be removed in cells infected, which leads to a source of recurrence of hepatitis c virus. Our previous experiments in vivo and in vitro showed that hepatitis c virus (HCV) core protein can activate specifically2’-5’ oligoadenylate synthetase (OAS) promoter regulated recombinant plasmid caspase3, thus leading to hepatic cell apoptosis infected by HCV. Interferon is normally used to participate in the treatment of hepatitis c. Japanese scholars Kerr found the2’-5’oligoadenylate synthetase can be found in the cell extract by using interferon as a stimulant. This study aims to clear and definite whether the interferon a-2b can activate specifically the OAS promoter regulated recombinant plasmid caspase3or not, and provide the experimental basis for its further application.Methods:(1)construct recombinant plasmid EGFP-OAS-re-caspase3, Use PCR, gel electrophoresis and PCR product sequencing to validate.(2) establish stable transfected cell lines:transfect EGFP-OAS-re-caspase3plasmid and EGFP plasmid to HL7702and HepG2cell lines,24h after transfection, observe and count the transfected efficiency in fluorescence microscope. G418maintains the selection until the cells express green fluorescent protein stably.(3) use flow cytometry, MTT, crystal violet staining and TUNEL to test and compare different transfected group HL7702cell and HepG2cell apoptosis rate under the action of interferon with different concentration.(4)nude mice tumor model:inject stable transfected HepG2/EGFP-OAS-re-caspase3and HepG2cells into nude mice subcutaneous, wait for tumor formation to inject the physiological concentration of interferon into tumor site, put death to animals after48hours and take the tumor. Use TUNEL technology to detect HepG2cells’ apoptosis rate.Results:(1)PCR, gel electrophoresis and sequencing results show that we successfully constructed recombinant plasmid EGFP-OAS-re-caspase-3.(2)24h after transfection, count transfected cells, which transfected efficiency is about70%under fluorescence microscope. visible cloning will be found after G418filters the cells for about2weeks, and the stable transfected cell lines will be established after one mouth.(3)The detection results of apoptosis under flow cytometry, MTT, crystal violet staining and TUNEL indicate:interferon a-2b can activate specifically the OAS promoter regulated recombinant plasmid caspase3,thus leading to the apoptosis of cells transfected by GV230/EGFP-OAS-re-caspase3,and the apoptosis rate of cells increases with the increase of the concentration of interferon(P<0.05); However, cells transfected by EGFP with0u/ml,100u/ml,500u/ml interferon a-2b, cell apoptosis rates are no statistically significant difference between groups (P>0.05);(4) nude mice tumor experiment results:the animal model is established successfully; the apoptosis rate of cells in HepG2/EGFP-OAS-re-caspase-3group is significantly higher than HepG2group (P<0.05).Conclusions:1.Interferon a-2b can specifically induces the apoptosis of hepatic cells transfected EGFP-OAS-re-caspase3plasmid in vitro and in vivo;2. the apoptosis rate of cells transfected by GV230/EGFP-OAS-re-caspase3increases with the increase of the concentration of interferon within a certain range;3. Interferon might strengthen the performance of OAS-re-caspase3to treat hepatitis c.