【摘要】 采用表达shRNA的载体构建了表达针对病毒HBsAgmRNA保守区的shRNA的质粒psiHBs,利用细胞模型和高压注射小鼠模型评价RNA干扰对HBV复制和基因表达的抑制作用。通过Western印迹检测细胞内的HBsAg,用ELISA检测细胞培养上清和血清中的HBsAg,采用Southern印迹检测HBV的复制中间体,最后通过免疫组化的方法检测肝组织切片中HBcAg的表达情况。结果显示pHBV1.3和psiHBs共转染HepG2后,与对照组相比病毒HBsAg和HBeAg的表达和病毒复制中间体的水平下降了90%以上,并且shRNA的作用效率存在序列特异性和剂量依赖性。在高压注射小鼠模型中,psiHBs表达的shRNA使小鼠血清中HBsAg的水平下降了80%以上,免疫组化检测显示,小鼠肝组织内HBcAg阳性细胞数减少了75.1%,而且shRNA的抑制作用至少能持续4d。研究显示载体表达的shRNA无论是在细胞或是在小鼠模型中都能对HBV的复制和基因的表达发挥序列特异性的抑制作用。本研究为我们下一步实现由RNAi介导的基因治疗提供了理论和技术支持。更多还原

【Abstract】 In this study,psiHBs was constructed through the backbone of psiRNA-vector to transcribe a small hairpin RNA(shRNA)corresponding to the messenger RNA of the HBV small surface antigen.After transfection of HepG2 cells or Balb/c mice with psiHBs and pHBV1.3,a plasmid containing 1.3-genomic-length replicative-competent HBV DNA,the viral antigens were evaluated by ELISA,Western blotting,or immunohistochemistry and the viral replicative intermediates were detected by Southern hybridization.The data indicated that after transfection of HepG2 cells with pHBV1.3 and psiHBs,the HBsAg and HBeAg levels were reduced by over 90%,and the replicative intermediates in cells were decreased to 10% relative to control.In hydrodynamic injection mouse model,Balb/c mice were co-injected with pHBV1.3 and psiHBs.The HBsAg levels in mice sera were reduced by 80% relative to the control group,whereas the number of HBcAg positive hepatocytes in the mouse liver sections was decreased substantially by 75.1%.Our study showed that shRNA inhibits the HBV replication and gene expression substantially and effectively.The RNAi-based approach might become a pentential and novel tool for medical therapeutics in the future.更多还原