丙型肝炎病毒体外感染胎肝细胞的初步研究

A preliminary study on hepatitis C virus infection of human fetal hepatocytes in vitro

目的 探索原代培养胎肝细胞对丙型肝炎病毒 (HCV)的易感性 ,旨在建立较为稳定实用的细胞感染模型。方法 感染血清与培养肝细胞共同孵育 6～ 8h后 ,收集不同时相点标本 ,用逆转录聚合酶链反应 (RT PCR)分别检测细胞内或上清液中正负链RNA ,免疫组化检测细胞内HCVNS3、NS5特异性抗原的表达情况 ,以及原位杂交检测细胞内HCV负链RNA。结果 接种感染血清 3d后 ,即可在细胞内或培养上清液中检出HCV正负链RNA ,间断检出至感染后第 17天。HCVNS3、NS5特异性抗原可在感染细胞内表达 ,阳性物质位于胞浆中。原位杂交法证实细胞内存在负链RNA ,也位于胞浆中。结论 原代培养的胎肝细胞对HCV不但易感 ,而且稳定地支持HCV复制。

Objective To establish a cell model of HCV infection, and to investigate the susceptibility of adult normal fetal hepatocytes to hepatitis C virus in primary culture. Methods Both the plus and minus strands of HCV RNA in infected cells and supernatant were examined by reverse transcription polymerase chain reaction (RT-PCR). The expression of HCV NS3,NS5 antigens in infected cells were detected by immunohistochemical assay with monoclonal antibodies. The minus strand of HCV RNA in infected cells were observed by in situ hybridization.Results ①The intracellular plus or minus strands of HCV RNA could be detected began on day 3 post-incubation and then detected intermittently until day 17 post-incubation in cells or supernatant. ② The positive signals of HCV NS3, NS5 antigens expressed within cytoplasm of infected cells.③ The minus strand of HCV RNA was located within cytoplasm.Conclusion Above results indicate that human fetal hepatocytes in primary culture are not only susceptible to HCV infection but can also maintain HCV replication stably.