Inhibition Of Hepatitis B Virus Expression In Mice By Rnai

Objective:Hepatitis B virus (HBV) infection is the crucial risk of chronic liver disease and hepatocellular carcinoma in humans. In this study, model was constructed by hydrodynamic tail vein injection method, meanwhile mice received shRNA vector. This interference may be foundation of advance research on immunological effect.Methods:1 Mice received HBV genome plasmid by hydrodynamic tail vein injection method and killed on the 4th day. We evaluated whether Model was successfully contructed by means of dose-response experiment, time-response experiment and three kinds of image systems.2 HBsAg and HBeAg in serum were assayed by time-resolved immunoflurometric assay in the clinical laboratory of Wuhan Tongji Hospital, Wuhan, Hubei, PR China. Total RNA was assayed by Realtime PCR. HBcAg in hepatocyte was measured by Immunohistochemistry.Results:1 Construction of mice model for HBV infectionMice received HBV genome plasmid by hydrodynamic tail vein injection method and killed on the 4th day.Results of HBsAg and HBeAg quantificationally assayerd showed that HBeAg gradually increased as more pBlue– HBV was injected. Meanwhile, GFP in hepatocyte was assayed by Flow cytometry that was expressed ?from day 2 to day 9. Image showed that there was green fluorescence in mice injected pGenesil– hk but not in normal mice. Therefore, such model was successful because there was a lot of HBsAg and HBeAg in serum and the expression was highest in day 3 to day 5 and lasted about 10days.2 effects of RNA interferenceResults of realtime PCR indicated that siRNA groups may inhibit the expression of HBsAg and HBeAg. Constructed siRNA could reduce HBV at mRNA level in vivo. Results of time-resolved immunoflurometric assay showed that HBsAg and HBeAg were significantly decreased in pGenesil– 1 and pGenesil– 2 groups and then at protein level, siRNA also effectively inhibited HBV in vivo. In immunohistochemistry experiments, there were lots of positive cells in control group but in pGenesil– 1 and pGenesil– 2 groups few positive cells were in a field. Therefore, siRNA may reduce expression of HBcAg in hepatocyte.Conclusion:In this model, constructed by hydrodynamic tail vein injection method, many HBsAg and HBeAg were expressed in liver and serum. Co-injected siRNA may effectively inhibit HBV and proteins related to it.Therefore, these results may be foundation of advance research on immunological effect.