基于HCV J6-JFH/Huh7.5.1抗HCV活性药物筛选体系的构建与验证  

Construction and Verification of Anti-HCV Drug Screening Technique Based on HCV J6-JFH / Huh7.5.1 Culture System

作　　者：岳蕾[1] 高凤平[1] 刘丽[1] 冯悦[1] 夏雪山[1,2] 

机构地区：[1]昆明理工大学生命科学与技术学院,云南昆明650500 [2]云南省分子医学研究中心,云南昆明650500

出　　处：《药物生物技术》2015年第4期288-292,共5页Pharmaceutical Biotechnology

基　　金：国家科技支撑计划项目(No.2014BAI01B01)

摘　　要：以J6-JFH/Huh7.5.1 HCV体外培养体系为基础,建立抗HCV体外药物筛选体系,并用环孢素A(Cs A)验证该体系的工作性能。用重组HCV(J6-JFH)感染Huh-7.5.1细胞,根据病毒增殖特征确定最适病毒感染复数;MTT法测定细胞毒性,得出CC50值;Real-time RT-PCR法测定HCV病毒载量,得出EC50值,计算药物的选择指数(SI),建立抗HCV药物筛选技术体系;用环孢素A验证该体系的工作性能。最适病毒感染复数为1;利巴韦林CC50=21.15μg/m L、EC50=2.256μg/m L和选择指数SI=9.375;干扰素EC50=0.194 IU/m L,用本技术体系测定Cs A的EC50值为(0.046±0.9)%。利巴韦林可作为合适的阳性药物对该技术体系进行质量控制;干扰素有稳定的抗病毒活性,但无细胞毒性,只能进行药物抗病毒活性质控。检测的Cs A的EC50值与已有的报道数据相似,并且Cs A抗HCV活性不受血清浓度的影响。结果证明,在2a型J6-JFH1/Huh7.5.1 HCV细胞培养体系基础上,成功建立了抗HCV药物筛选体系,该技术体系可行并稳定,为抗HCV药物筛选提供了良好的技术平台。Based on J6-JFH / Huh7. 5. 1 hepatitis C virus culture system in vitro,the anti-HCV drug screening technique was established,and its working performance was further tested by Cyclosporine A（ Cs A）. Huh-7. 5. 1 cells were infected by recombinant HCV virus（ J6-JFH）,virus’ optimal multiplicity of infection（ MOI） was determined according to the viral proliferation characteristics.MTT assay was used to detect cytotoxicity and to,calculate CC50 value. Real-time RT-PCR assay was used to quantitatively determine the HCV viral load,and to calculate EC50 value. The virus’ optima multiplicity of infection（ MOI） was 1. Ribavirin CC50= 21. 15μg /m L and,EC50= 2. 256 μg / m L,the selection index SI = 9. 375. Interferon EC50= 0. 194 IU / m L. Furthermore,Cs A was taken as the candidate compound to test the working performance of this technique. It was showed that the EC50 values of Cs A were（ 0. 046 ±0. 9%） μmol / L. Moreover,the anti-HCV activity of Cs A hasn’t been affected by the serum concentration. Ribavirin can be used as a suitable positive drug for quality control on this technical system. However,interferon has a stable antiviral activity but non-cytotoxic.It could be used as quality control to evaluate drugs’ antiviral activity. It was showed that the EC50 values of Cs A cove similar to previous reports. Moreover,the anti-HCV activity of Cs A hasn’ t been affected by the serum concentration. The results verified that the anti-HCV drug screening technique system which was based on the HCV genotype 2a J6-JFH / Huh7. 5. 1 cell culture was successfully established. And the system is a suitable technique for anti-HCV drug screening. So a useful technique platform for anti-HCV drug screening was provided.

关 键 词：Huh 7.5.1 HCV 环孢素A 药物筛选 人血清 抗病毒活性 

分 类 号：R575.1[医药卫生—消化系统]