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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 a self-replicating hepatitis C virus (HCV) in vitro cell infection model. Methods HCV RNA transcripts of HCV-FL-J6JFH and negative control FL-J6JFH (GND) were prepared and transfected into Huh-7.5 cells respectively. HCV RNA levels were detected by FQ-PCR and IFA Intracellular HCV protein expression. Na 觙 ve Huh-7.5 cells were infected with the transfected cell supernatant and the production of HCV infectious virus particles (HCVcc) was examined. The cells were treated with different concentrations of IFNa and the sensitivity of the established cellular infection model to IFNa was observed. Results On the fifth day after transfection, the HCV RNA in FL-J6JFH transfected cells was 1.2 × 106 GE / μg RNA, which dropped to the lowest level on the 9th day and then rose to reach the highest value of 6.5 × 106GE / μg RNA on the 13th day. From then on, until 59th day, both of them remained at about 106GE / μg RNA, while HCV RNA in FL-J6JFH (GND) transfected cells disappeared rapidly. HCV protein expression was always detected in FL-J6JFH transfected cells, but not in control cells. From day 5 onward, HCVcc was produced in FL-J6JFH transfected cells at each time point. IFNα can effectively inhibit HCVcc infection Huh-7.5 cells in a dose-dependent manner. Conclusion HCV2a-type in vitro cell infection model has been successfully established. IFNα can inhibit HCV RNA replication in HCV-infected FL-J6JFH cells, providing an experimental platform for studying the structure and function of HCV.