丙型肝炎阳性血清直接感染HepG2细胞的初步研究  

Primary study of HCV replication in HepG2 cells inoculated by anti-HCV positive and HCVRNA RT-PCR positive serum

作　　者：赖力英[1] 龚国忠 曾庆善[1] 郑宣鹤 张淑安 

机构地区：[1]第二附属医院,长沙410011

出　　处：《湖南医科大学学报》2000年第6期548-552,共5页Bulletin of Hunan Medical University

基　　金：国家自然科学基金资助项目! (396 70 6 7)

摘　　要：目的 :用丙型肝炎 (丙肝 )阳性血清 (抗HCV阳性及HCVRNART PCR阳性 )感染HepG2细胞 ,以期建立稳定的HCV感染的细胞模型。方法 :采用丙肝阳性血清连续感染法感染HepG2细胞 ,用逆转录多聚酶链反应检测传代HepG2细胞内HCVRNA正链和负链 ,电镜观察感染后的细胞内的病毒颗粒 ,免疫组化方法检测感染后的细胞内丙肝病毒蛋白抗原 (HCVNS5、HCVcapsid)的存在。结果 :感染后传代的第一代至第三代细胞内检出HCVRNA正链及负链 ;在感染后传代的细胞内发现球形、密集分布的类丙肝病毒颗粒 ,直径为 30～ 6 0nm ;免疫金银染色发现部分细胞内有聚集的银染颗粒 ;免疫电镜检查在细胞胞浆的囊泡样结构中发现聚集的金颗粒。结论 :HCV可在HepG2细胞内复制 ,可形成病毒颗粒 ,可随细胞传代。Objective: To study HCV replication in HepG2 cell line and to establish a stable HepG2 cell line infected with HCV. Methods: Continous inoculation of anti HVC positive and HCVRNA RT PCR positive sera was used to infect HepG2 cells. The plus and minus strains of HCVRNA were detected by reverse transcription polymerase chain reaction in the HepG2 cells. Virus particles were observed in postinoculated HepG2 cells by transmission elctron microscope. Immunohistochemical staining was used respectively to detect HCV antigen, HCVNS5 and HVC capsid in postinoculated HepG2 cells. Results: HCV RNAs (both plus and minus strains) were found from the first passage to the third passage of postinoculation HepG2 cells, and HCV viruslike particles were detected in the third passage of the infected HepG2 cells, those particles mostly occurred within cytoplasm, their diameters were between 30～60?nm, and gathering silver particles were found in immunogold silver staining of HCV NS5/capsid protein in infected HepG2 cells. Gathering gold particles were found mostly within vesicles in cytoplasm by immuno gold electron microscopy. Conclusion: HepG2 cell line infected by HCV is established by continuous inoculation of anti HCV positive and HCVRNA positive sera. HCV can replicate and produce virus like particles in HepG2 cells, which may occur in the serial passages of post infected HepG2 cells. [

关 键 词：HEPG2细胞 免疫组化 丙型肝炎 HCV 

分 类 号：R512.6[医药卫生—内科学]