丙型肝炎病毒阳性血清体外感染人肝细胞的研究

目的 研究丙型肝炎病毒(HCV)抗体(Ab)阴性,HCV-RNA阳性血清建立体外感染肝细胞模型.方法 HCV Ab阴性,HCV-RNA阳性的窗口期血清与人肝细胞共同培养,用反转录-聚合酶链反应(RT-PCR)、免疫荧光染色、Western blot、共聚焦显微镜和透射电镜等方法检测细胞内HCV核酸复制、蛋白质表达及超微结构改变.结果 细胞与病毒共同培养7～45 d,细胞内和/或培养上清中可间断检出HCV正、负链RNA;细胞浆内有HCV 核心和NS3抗原的表达;细胞超微结构有改变,并于感染后第24天时观察到类似病毒样颗粒.结论 窗口期血清中的HCV能在人肝细胞7701中复制一段时间.

Study on hepatocyte cell infected in vitro by hepatitis C virus positive serum

Objective To study the hepatocyte cells infected by hepatitis C virus (HCV) positive serum. Methods Human hepatocyte 7701 was incubated with HCV RNA-positive and HCV antibody(Ab) negative sera BP52. Then, the expression of HCV antigen and the presence of HCV-RNA in cell and supernatant were assayed by RT-PCR, sequence analysis, immunofluorescent staining, Western blot, confocal laser microscopy. The ultrastructural changes of infected cells were observed by electro-microscopy. Results Plus-strand RNA and minus-strand RNA were intermittently detected in cell and/or supernatant on day 7-45 after infection. Sequence analysis demonstrated that the positive DNA nucleic acids were identified with HCV 5′-non-coding region(NCR) sequence. HCV core and NS3 protein were expressed in cytoplasm of infected cells. After 2 or 3 weeks, obvious intracellular ultrastructural changes and virus-like particles were observed. Conclusion human hepatocyte 7701 could support replication of HCV in vitro, which could be a useful tool for setting up cell model of HCV infection and studying the mechanism of HCV infection.