【摘要】 为了研究由pRNA携带的siRNA(HBVsi18-42)所介导的RNAi过程能有效地抑制HBV的基因表达和病毒复制,我们利用细胞模型和高压注射小鼠模型评价HBVsi18-42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含量,用ELISA检测细胞培养上清和小鼠血清中的HBsAg水平,采用Southern印迹检测HBV的复制中间体,通过免疫组织化学检测肝组织切片中HBcAg的表达情况。试验结果显示,HBVsi18-42能以剂量依赖的方式在293T细胞中抑制HBsAg的表达以及在HepG2细胞中下调病毒HBsAg和HBeAg的表达和病毒复制中间体的水平。在小鼠模型中,注射后的3d内HBVsi18-42使小鼠血清中HBsAg的水平分别下降了98.98%、77.07%和60.73%,免疫组织化学检测显示,在注射后的第3天小鼠肝组织内HBcAg阳性细胞数减少了79.1%。初步结果显示HBVsi18-42无论是在细胞或是在小鼠模型中都能下调HBV的复制和基因的表达。本研究为我们下一步实现由pRNA介导的靶向RNAi及基因治疗提供了理论和技术支持。更多还原

【Abstract】 The objective of our present study is to explore the potential use of pRNA as a bio-carrier of siRNA to inhibit HBV gene expression and replication. After co-transfected with pHA-HBs into 293T cells, HBVsi18-42, a pRNA-escorted siRNA, suppressed HBsAg and accumulated in the cells in a dose-dependent fashion. HBVsi18-42 substantially inhibited HBV gene expression and replication initiated by pHBV1.3 in HepG2 cells. In hydrodynamic injection mouse model, Balb/cJ mice were co-injected with pHBV1.3 and HBVsi18-42. Serum concentrations of HBsAg were analyzed by ELISA on days 1, 2, and 3 post-injection. HBV core protein in mouse liver was visualized by immunohistochemical staining. The results showed that the HBsAg levels in mice sera were reduced by 60%~90% in consecutive days relative to the control group, and the number of HBcAg positive hepatocytes in the mouse liver sections was decreased substantially by 79.1%. Our preliminary data showed that pRNA could be used as bio-carrier for the delivery of siRNA to knock down HBV gene expression and repress viral replication.更多还原