【摘 要】
：
目的 通过建立拉米夫定耐药乙型肝炎病毒体外细胞模型,评价盐酸千金藤碱(cepharanthine hydrochloride,CH)对拉米夫定耐药乙型肝炎病毒复制与表达的体外抑制作用.方法 首先从拉米夫定耐药的慢性乙型肝炎患者血清中提取HBV DNA,进一步通过PCR技术扩增出HBV全基因.然后将该HBV全基因定向克隆至pHY106新型真核表达载体,通过细胞转染技术建立拉米夫定耐药乙型肝炎病毒体外
【机 构】
：
河南省(郑州大学)医药科学研究院,河南郑州450052;河南省肝病药理重点实验室,河南郑州450052;郑州大学药学院,河南郑州450001
论文部分内容阅读
目的 通过建立拉米夫定耐药乙型肝炎病毒体外细胞模型,评价盐酸千金藤碱(cepharanthine hydrochloride,CH)对拉米夫定耐药乙型肝炎病毒复制与表达的体外抑制作用.方法 首先从拉米夫定耐药的慢性乙型肝炎患者血清中提取HBV DNA,进一步通过PCR技术扩增出HBV全基因.然后将该HBV全基因定向克隆至pHY106新型真核表达载体,通过细胞转染技术建立拉米夫定耐药乙型肝炎病毒体外细胞模型.将不同浓度的CH加入细胞上清液,每2d更换一次培养液,药物作用6d后,收集细胞培养上清液及细胞.ELISA检测药物作用后细胞上清液中HBeAg与HbsAg的变化,荧光定量PCR方法检测细胞内HBV DNA含量的变化.为了进一步探讨CH抗HBV的作用机制,本研究应用反转录聚合酶链反应(RT-PCR)方法检测了药物作用后细胞内宿主热应激蛋白HSc70 mRNA的变化.结果 CH作用6d后,细胞上清液中HBeAg的表达明显减少,同时细胞内HBV DNA的含量也呈浓度依赖性的降低.CH能够显著降低细胞内HSc70 mRNA的表达水平,且与细胞上清液中HBeAg及细胞内HBV DNA的含量的下降有明显的相关性.结论 CH能够有效抑制拉米夫定耐药乙型肝炎病毒的复制与表达,抑制宿主细胞HSc70的表达可能为其发挥抗HBV作用机制之一.
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