Chronic hepatitis B is a primary cause of liver-related death. Interferon alpha (IFN-alpha) is able to inhibit the replication of hepadnavirus, and the sustained and stable expression of IFN-alpha at appropriate level may be beneficial to HBV clearance. With the development of molecular cloning technology, gene therapy plays a more and more important role in clinical practice. In light of the findings, an attempt to investigate the anti-HBV effects mediated by a eukaryotic expression plasmid (pSecTagB-IFN-alpha) in vitro was carried out.
Methods
HBV positive cell line HepG2.2.15 and its parental cell HepG2 were transfected with pSecTagB-IFN-alpha or empty plasmid by using LipofectamineTM 2000 reagent. The expression levels of IFN-alpha were determined by reverse transcriptase polymerase chain reaction (RT-PCR) and ELISA methods. The effects of pSecTagB-IFN-alpha on HBV mRNA, DNA and antigens were analyzed by real-time fluorescence quantitative PCR (qRT-PCR) and ELISA assays. RT-PCR, qRT-PCR and western blot were employed to investigate the influence of pSecTagB-IFN-alpha on IFN-alpha-induced signal pathway. Furthermore, through qRT-PCR and ELISA assays, the suppressive effects of endogenously expressed IFN-alpha and the combination with lamivudine on HBV were also examined.
Results
pSecTagB-IFN-alpha could express efficiently in hepatoma cells, and then inhibited HBV replication, characterized by the decrease of HBV S gene (HBs) and HBV C gene (HBc) mRNA, the reduction of HBV DNA load, and the low contents of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). Mechanism research showed that the activation of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signal pathway, the up-regulation of IFN-alpha-induced antiviral effectors and double-stranded (ds) RNA sensing receptors by delivering pSecTagB-IFN-alpha, could be responsible for these phenomena. Furthermore, pSecTagB-IFN-alpha vector revealed effectively anti-HBV effect than exogenously added IFN-alpha. Moreover, lamivudine combined with endogenously expressed IFN-alpha exhibited stronger anti-HBV effect than with exogenous IFN-alpha.
Conclusion
Our results showed that endogenously expressed IFN-alpha can effectively and persistently inhibit HBV replication in HBV infected cells. These observations opened a promising way to design new antiviral genetic engineering drugs based on IFN-alpha. 作者: StephenW 时间: 2013-8-31 16:49