HCV 2a J6JFH细胞感染模型的建立及初步应用  

Establishment and Preliminary Application of Cell Culture Model of HCV 2a FL-J6JFH Strain

作　　者：任浩[1] 王永智[1] 赵平[1] 刘媛[1] 戚中田[1] 

机构地区：[1]第二军医大学微生物学教研室上海市医学生物防护重点实验室,上海200433

出　　处：《中国生物制品学杂志》2009年第1期1-5,9,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金(30600529;30872247);军队"十一五"专项课题基金(06H021;06Z027);上海市重点学科建设项目(B901)

摘　　要：目的建立能够自主复制的丙型肝炎病毒(HCV)体外细胞感染模型。方法制备HCV2a FL-J6JFH和阴性对照FL-J6JFH(GND)RNA转录体,分别转染Huh-7.5细胞,荧光定量PCR(FQ-PCR)检测细胞内HCV RNA水平,免疫荧光法(IFA)检测细胞内HCV蛋白的表达。以转染细胞上清液感染Na觙ve Huh-7.5细胞,检测HCV感染性病毒颗粒(HCVcc)的产生。将细胞用不同浓度的IFNα处理,观察所建立的细胞感染模型对IFNα的敏感性。结果转染后第5天,FL-J6JFH转染细胞内HCV RNA为1.2×106GE/μg RNA,第9天下降至最低水平,随后上升,于第13天达到最高值6.5×106GE/μg RNA,此后直至第59天,均保持在106GE/μg RNA左右,而FL-J6JFH(GND)转染细胞内HCV RNA则很快消失。FL-J6JFH转染的细胞内始终可以检测到HCV蛋白表达,而对照细胞内均未检测到。从第5天起,各时间点的FL-J6JFH转染细胞均能产生HCVcc。IFNα能够有效抑制HCVcc感染Huh-7.5细胞,并呈剂量依赖性。结论已成功建立了能持续产生HCVcc的HCV2a型体外细胞感染模型,IFNα能抑制FL-J6JFH HCVcc感染细胞中HCV RNA的复制,为研究HCV的结构与功能提供了实验平台。Objective To establish the autonomously replicating in vitro cell culture model of hepatitis C virus （HCV）. Methods The RNA transcripts of intragenotypic 2a / 2a genome FL-J6JFH and the replication-defective NS5B negative control FL-J6JFH （GND） were transfected into Huh-7. 5 cells respectively. At the indicated time points, total RNAs were isolated from the transfected Huh-7.5 cells, and the levels of HCV RNA were determined by HCV-specific fluorescent quantitative（FQ） RT-PCR. The expression of HCV protein was determined by immunofluorescence staining. Naive Huh-7. 5 ceils were inoculated with the supernatants collected at various time points posttransfection to determine the production of cell culture produced HCV（HCVcc）. In addition, naive Huh-7. 5 cells were treated with IFNα at various concentrations to evaluate the sensitivity of established infectious cell culture model to IFNoc Results The HCV RNA level in Huh-7.5 cells on day 5 after transfection was 1.2 × 10^6 GE/μg RNA, and decreased to a minimum level on day 9, then increased gradually and reached a maximum level of 6.5 × 10^6 GE/μg RNA on day 13, which was maintained at 10^6 GE/μg RNA until day 59. However, the HCV RNA in Huh-7. 5 cells transfected with FL-J6JFH（GND） disappeared rapidly. HCV proteins were only detected in the Huh-7. 5 cells transfected with FL-J6JFH RNA. HCVcc were observed in Huh-7. 5 cells collected at various time points from day 5 after transfection with FL-J6JFH. IFNα showed a dose-dependent inhibitory effect on the infection of HCVcc to Huh-7. 5 cells. Conclusion An in vitro cell culture model of HCV 2a FL-J6JFH, producing HCVcc continuously, was successfully established, and IFNα inhibited the replication of HCV RNA in HCVcc-infected cells, which provided a useful platform for studying the structure and function of HCV.

关 键 词：丙型肝炎病毒 2a基因型 J6JFH 细胞感染模型 

分 类 号：R512.60[医药卫生—内科学]