Xin Xiao-min, Li Gui-qiu, Guan Xiu-ru, Li Di, Xu Wei-zhen, Jin Ying-yu, Gu Hong-xi
Department of Microbiology, Harbin Medical University, Harbin 150081,Heilongjiang Province, China.
Hepatogastroenterology. 2008 Nov-Dec;55(88):2178-83.
BACKGROUND/AIMS: Hepatitis B virus (HBV) infection is a world-wide health problem. The major obstacles for current anti-HBV therapy are the low efficacy and the occurrence of drug resistant HBV mutations. Recent studies have demonstrated that combination therapy can enhance antiviral efficacy and overcome the shortcomings. Here, the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of HBV nuclear localization signal (NLS) was monitored in HepG2.2.15 cells.
Recombinant plasmid psil-HBV was constructed and transfected into HepG2.2.15 cells. At 48, 72 and 96 h after transfection, culture media were collected and cells were harvested for HBV replication assay. HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. Intracellular viral DNA and covalently closed circular DNA (cccDNA) was quantified by real-time PCR. HBV viral mRNA was reverse transcribed and quantified by reverse-transcript PCR.
Our data demonstrated that three used siRNAs showed marked anti-HBV effects. The expression of HBsAg and the replication of HBV DNA could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, combination of siRNAs, compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication, even though the final concentration of siRNA in the therapy was the same. More importantly, we showed that combination therapy significantly suppressed HBV cccDNA amplification.
Our results revealed that combination of siRNAs mediated a stronger inhibition on viral replication and antigen expression in HepG2.2.15 cells, especially, the amplification of cccDNA.
背景/目的：乙型肝炎病毒（HBV）感染是一个全球性的健康问题。当前抗HBV治疗的主要障碍是疗效低下和耐药性HBV突变的发生。最近的研究表明，联合治疗可以提高抗病毒疗效并克服这些缺点。在此，在HepG2.2.15细胞中监测了靶向HBV核定位信号（NLS）不同位点的小干扰RNA（siRNA）组合介导的抑制作用。
构建重组质粒psil-HBV并转染到HepG2.2.15细胞中。转染后48、72和96小时，收集培养基并收获细胞用于HBV复制测定。通过酶联免疫吸附测定法检测细胞培养基中的HBsAg和HBeAg。通过实时PCR对细胞内病毒DNA和共价闭合环状DNA（cccDNA）进行定量。对HBV病毒mRNA进行逆转录并通过逆转录PCR进行定量。
我们的数据表明，所使用的三种siRNA显示出显著的抗HBV作用。siRNA可以以剂量依赖的方式特异性抑制HBsAg的表达和HBV DNA的复制。此外，与单独使用每种siRNA相比，siRNA组合对抗原表达和病毒复制具有更强的抑制作用，即使治疗中siRNA的最终浓度相同。更重要的是，我们表明联合治疗显著抑制了HBV cccDNA的扩增。
我们的结果表明，siRNA组合在HepG2.2.15细胞中介导了对病毒复制和抗原表达更强的抑制作用，尤其是对cccDNA的扩增。
