人肝癌细胞株7721HCV体外感染模型的建立

目的建立接近体内自然感染状态并能稳定支持HCV体外长期复制的感染细胞模型。方法将慢性丙型肝炎患者的血清与人肝癌细胞系7721共同孵育8小时后,分别以反转录-聚合酶链反应、免疫组化、原位杂交检测细胞和/或培养上清中的HCV正、负RNA、HCV抗原的表达及HCV-RNA在感染细胞内的定位。结果感染血清和细胞共同孵育后的第2～65天,从细胞内和培养上清中均可间断地检测出HCV正、负RNA,即使其间细胞传代4次HCV NS_3抗原、NS_5抗原在细胞内能稳定表达,原位杂交显示HCV RNA阳性物质多位于细胞浆。结论 7721细胞不但对HCV易感,而且可以稳定地支持HCV的体外长期复制,此模型可以用于HCV感染、复制机理的研究、抗病毒药物的评价及保护性抗体/疫苗的初步筛选。

Establishment of HCV Infection Model in Vitro Using a Human Liver Cancer Cell Line 7721

Objective To establish an infected cell model that is close to the HCV replication in vivo and can support HCV long-term replication in vitro. Method A human liver cancer cell line 7721 was tested for its susceptibility to HCV by cultivation with a serum from chronic hepatitis C patient for 8 houres. After inoculation, plus-and minus-strand of HCV RNA, the expression of HCV NS_3,NS_5 antigen and the location of HCV-RNA in cell and/or supernatant were examined by RT-PCR, immunohistochemistry and in situ hybridization, respectively. Results It was found that plus-and minus strand of HCV RNA could be discontinuously detected in both cell can supernatant over a period of 64 days postinoculation, even if the 7721 cell line had been subcultured for 4 times. HCV NS_3, NS_5 antigen could be expressed in cells and HCV-RNA was mainly located within cytoplasm. Conlusion The results suggested that 7721 cell line was not only susceptible to HCV, but also could support its long-term replication in vitro. This HCV replication model in vitro was a useful tool in the study on mechanism of HCV infection and replication, the evaluation of antiviral compounds, the primary selection of neutralization assays and HCV vaccine development.