Yu Haotian, Hou Zhaohua, Han Qiuju, Zhang Cai, Zhang Jian
Institute of Immunopharmacology and Immunotherapy, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua West Road, Jinan 250012, China.
Virol J. 2013 Aug 29;10:270. doi: 10.1186/1743-422X-10-270.
Chronic hepatitis B is a primary cause of liver-related death. Interferon alpha (IFN-α) is able to inhibit the replication of hepadnavirus, and the sustained and stable expression of IFN-α 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-α) in vitro was carried out.
HBV positive cell line HepG2.2.15 and its parental cell HepG2 were transfected with pSecTagB-IFN-α or empty plasmid by using Lipofectamine™ 2000 reagent. The expression levels of IFN-α were determined by reverse transcriptase polymerase chain reaction (RT-PCR) and ELISA methods. The effects of pSecTagB-IFN-α 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-α on IFN-α-induced signal pathway. Furthermore, through qRT-PCR and ELISA assays, the suppressive effects of endogenously expressed IFN-α and the combination with lamivudine on HBV were also examined.
pSecTagB-IFN-α 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-α-induced antiviral effectors and double-stranded (ds) RNA sensing receptors by delivering pSecTagB-IFN-α, could be responsible for these phenomena. Furthermore, pSecTagB-IFN-α vector revealed effectively anti-HBV effect than exogenously added IFN-α. Moreover, lamivudine combined with endogenously expressed IFN-α exhibited stronger anti-HBV effect than with exogenous IFN-α.
Our results showed that endogenously expressed IFN-α 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-α.
慢性乙型肝炎是肝脏相关死亡的主要原因。α干扰素（IFN-α）能够抑制嗜肝DNA病毒的复制，在适当水平持续稳定表达IFN-α可能有利于乙肝病毒的清除。随着分子克隆技术的发展，基因治疗在临床实践中发挥着越来越重要的作用。基于这些发现，我们尝试在体外研究真核表达质粒（pSecTagB-IFN-α）介导的抗乙肝病毒作用。
采用Lipofectamine™ 2000试剂将pSecTagB-IFN-α或空质粒转染乙肝病毒阳性细胞系HepG2.2.15及其亲本细胞HepG2。通过逆转录聚合酶链反应（RT-PCR）和酶联免疫吸附测定（ELISA）方法测定IFN-α的表达水平。采用实时荧光定量PCR（qRT-PCR）和ELISA检测pSecTagB-IFN-α对乙肝病毒mRNA、DNA和抗原的影响。运用RT-PCR、qRT-PCR和蛋白质免疫印迹法研究pSecTagB-IFN-α对IFN-α诱导信号通路的影响。此外，通过qRT-PCR和ELISA检测内源性表达的IFN-α以及与拉米夫定联合使用对乙肝病毒的抑制作用。
pSecTagB-IFN-α能够在肝癌细胞中高效表达，进而抑制乙肝病毒复制，表现为乙肝病毒S基因（HBs）和乙肝病毒C基因（HBc）mRNA水平降低、乙肝病毒DNA载量减少以及乙肝表面抗原（HBsAg）和乙肝e抗原（HBeAg）含量降低。机制研究表明，通过递送pSecTagB-IFN-α激活Janus激酶（JAK）-信号转导及转录激活因子（STAT）信号通路、上调IFN-α诱导的抗病毒效应分子和双链（ds）RNA传感受体，可能是导致这些现象的原因。此外，pSecTagB-IFN-α载体显示出比外源性添加的IFN-α更有效的抗乙肝病毒作用。而且，拉米夫定与内源性表达的IFN-α联合使用比与外源性IFN-α联合使用表现出更强的抗乙肝病毒作用。
我们的结果表明，内源性表达的IFN-α能够有效且持续地抑制乙肝病毒感染细胞中的乙肝病毒复制。这些观察结果为基于IFN-α设计新型抗病毒基因工程药物开辟了一条有前景的途径。
