Antigene Locked Nucleic Acid Inhibits Hepatitis B Virus Coding Chain C Gene Expression In Transgenic Mice

Chapter one Design,screening and identification of HBV C coding chain antigene locked nucleic acidObjective: We designed and synthesized locked nucleic acid(LNA)fragments for hepatitis B virus(HBV)coding chain C gene,selected HBV transgenic mice as the research object,and and screened out the best dosage,route and mode of administration.Methods: According to the 2404~2418 nt site of the C gene of the hepatitis B virus coding chain,RNAstructure 5.0 software was used to design and synthesize the anti-gene LNA fragment 5?-CGA*CGCGGCGA*T*TGA*-3?,HBV transgenic mice were devided into different groups according to different dosage,route and mode of administration,and then transfected by LNA under the mediated effect of cationic polymer,orbital venous blood was collected before administration,1,3,5 and 7 days after administration.serum HBV DNA was detected by PCR fluorescent probe method,HBs Ag and HBe Ag were detected by magnetic particle chemiluminescence method.Single strand specific endonuclease digestion experiment was used to identify the stability of LNA fragments.Results: On the 7th day after administration,the 0.5 ?g/g dose group,the tail vein injection group and the 1/48 h administration group had significant inhibitory effects on viral HBV DNA replication,HBs Ag and HBe Ag expression.HBV DNA replication inhibition rates were 57.09%,56.61% and 57.22%,respectively.HBs Ag expression inhibition rates were49.29%,48.65% and 48.41%,HBe Ag were 71.72%,69.95% and 69.44%,respectively.LNA had strong resistance to nuclease degradation.Conclusion: The optimal dosage of LNA fragment 5?-CGA*CGCGGCGA*T*TGA*-3?is 0.5 ?g/g,the route of administration was tail vein injection,and the way of administration was once / 48 h.Chapter two Antigene locked nucleic acid inhibits hepatitis b virus coding chain c gene expression in transgenic miceObjective: To investigate the antiviral effect of anti-gene LNA targeting the 2404~2418 nt site of the C gene of the coding chain of HBV in HBV transgenic mice.Methods: Thirty HBV transgenic mice were completely randomly divided into 5 groups,with6 mice in each group,namely 5% GLU blank control group,irrelevant sequence control group,lamivudine control group,antisense LNA control group and anti-gene group.The 5% GLU blank control group,the irrelevant sequence control group,the antisense LNA control group and the anti-gene LNA experimental group were adminstrated by tail vein injection,and the lamivudine control group was administered by continuous gastrogavage.orbital venous blood was collected before administration,1,3,5 and 7 days after administration.serum HBV DNA was detected by PCR fluorescent probe method,HBs Ag and HBe Ag were detected by magnetic particle chemiluminescence method.The expression level of HBV C-m RNA,HBs Ag and HBs Ag in liver were detedted by immunohistochemical technique.The influence of anti-gene LNA on hepatorenal cell structure and function were measured by HE staining,blood routine test and biochemical markers test.Results: On the 1st,3rd,5th and 7th days after administration,the anti-gene LNA group had significant inhibitory effects on HBV DNA replication and HBs Ag and HBe Ag expression.HBV DNA replication inhibition rates were 20.68%,45.66%,52.33%,and 55.68%,respectively.HBs Ag expression inhibition rates were 20.44%,36.17%,44.90% and 47.87%,respectively.HBe Ag were 35.65%,54.29%,65.08% and 72.67%,respectively.Compared with the control group,the difference was statistically significant,P < 0.05,the relative expression of HBV C m RNA in the liver and the positive cell rates of HBs Ag and HBcAg were 0.36,38.20% and 32.50%,respectively.Compared with the control group,the differencewas statistically significant,P < 0.05.The results of HE staining,blood routine test and biochemical markers test showed that there was no significant influence of anti-gene LNA on hepatorenal cell structure and function.Conclusion: The 2404~2418 nt sites of C gene of hepatitis B virus coding chain are effective targets for anti-gene LNA therapy.