In Vitro Study On Inhibition Of HBV Expression By Antisense Locked Nucleic Acid Directed Against HBV S Gene

Objective:To investigate the inhibition of HBV expression in vitro by cationic liposome-mediated antisense locked nucleic acid directed against the translational initiation of S gene and to evaluate the anti-HBV effect of LNA.Methods:1. Identification of HepG2.2.15 cells: the functional activity and subtye of HepG2.2.15 HBV were identified by PCR,ELISA and immunochemistry straining.2. Determination of the dose of cationic liposome and the transfection efficiency of HepG2.2.15 cells:The cationic liposomes with concentrations of 2.5, 5.0,7.5, 10.0μl/ml were used to transfecting HepG2.2.15 cells respectively, and the culture media were collected for determination of the levels of HBsAg and HBeAg by ELISA. The cytoxicity of cationic liposomes on cells was detected by MTT and cell apoptosis by apoptosis detection kit. After 48 hours of transfection of HepG2.2.15 cells.With the four different concentrations of cationic liposome-mediated pEGFP-N1 the quantity of green fluorescent protein expressde were used to determine the susceptibility and transfection efficiency.3. ASODN design:ASODNs directed against HBV S translation initiator were designed and synthesized. They were modified differently by LNA, completement phosphorothioate, natural sequence respectively, Non-completementary sequence was used as control. 4. Optimization of the transfection parameters for transfecting PSODN to HepG2.2.15 cells mediated by cationic liposome.Transfections were performed with 5μl/ml and 7. 5μl /ml of cationic liposome respectively. The cytoxicity of PSODN/liposomes on cell was detected by MTT and cell apoptosis by apoptosis detection kit.5. Detection of LNA stability of anti-ribozyme in vitro:LNA, LNA/liposome, PSODN and PSODN/liposomes were digested by S1 Nuclease, then the digestive products were observed by electrophoresis, according to which the stability of modified PSODN and PSODN/liposomes ware determined.6.Detection of antiviral inhibitory rate of LNA: LNA/liposomes, natural sequence/ liposomes, Non-completementary sequence/liposome, and Lamivudine were added to HepG2.2.15 cells co-culture.The cells without any sequence and liposomes group were taken as negative control. Cells supernatant were collected every 48h. The dynamic change of HBsAg and HBeAg in supernatant were detected by ELISA and also that of HBV DNA by fluorescence quantitative PCR, for 10 days continously.7. The cytoxicity of LNA/liposomes on cells was detected by MTT and cell apoptosis was detected by apoptosis detection kit.Results:1.The results show that HepG2.2.15 cells were able to consecutively secrete HBsAg,HBeAg and Dames Particles. HBV DNA in HepG2.2.15 cells supernatant was positive and HBV DNA in cells was subtype ayw by PCR;HBsAg was positive in cells by immunochemistry staining.2. The doses of cationic liposome should be less than 7.5μl/ml for transfection; cationic lipid could inhibit the secretion of HBsAg and HBeAg. Cationic liposomes have certain toxic effects (compared with control group P <0.05) on cells, it could induce cell apoptosis. HepG2.2.15 cells transfected with plasmid pEGFP-N1 by cationic liposome could express green fluorescent protein, but the transfection efficiency was low(less than 23%)3. The optimal proportion of ASODN/ liposome is 3μg/5μl/ml. If the proportion was higher , there would be adverse effect on cells growth.4. ASODN modified by LNA has good stability of anti-enzyme digestion in vitro but both PSODN and ASODN/ liposome are unstable for anti-enzyme digestion in vitro.5.The ASODNs directed against HBV S gene could inhibite secreting of HBsAg and HBeAg (with control group P <0.05), the inhibition rates of HBsAg and HBeAg in LNA group were up to 67.7% and 59.7% respectively, which were better than in the groups of ASODNs and lamivudine. FQ-PCR results showed that HBV DNA was positive in cells supernatants, the HBV DNA inhibition rates of LNA were up to 63.3% (compared with control group P <0.05) ,which was consistent with ELISA results.6. The cytotoxicity of LNA/liposome is on host cells is lower comparing with liposomes control group, there was no significant difference. LNA also can induce cell apoptosis.Conclusion:1. HepG2.2.15 cells have excellent HBV expression activity. It could be taken as a good model for anti-HBV medicine research.2.Cationic liposomes could inhibit the expression of HBsAg and HBeAg in HepG2.2.15 cells and have toxicity on cells somewhat and also could induce apoptposis. HepG2.2.15 cells were sensitive to cationic liposome-mediated gene transfection.3. ASODN modified by LNA has good stability of anti-enzyme digestion.4. Antisense LNA directed against HBV S gene could effectively inhibit the expression of HBsAg, HBeAg and HBV gene. The inhibition rates were higher than ASODNs and lamivudine group. LNA/liposomes mixture has low cytotoxicity on host cells.