干扰素刺激基因ISG20抗丙型肝炎病毒的作用研究  

Anfiviral activities of ISG20 against hepatitis C virus

作　　者：徐华[1] 雷宇[1] 钟珊[1] 彭凤英[2] 周智[1] 李奎 任红[1] 

机构地区：[1]重庆医科大学附属第二医院感染病科重庆市肝病治疗研究中心,400010 [2]重庆医科大学附属第一医院传染科 [3]Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN, USA

出　　处：《中华肝脏病杂志》2013年第1期33-37,共5页Chinese Journal of Hepatology

基　　金：国家自然科学基金（81171563、31000397）l重庆市科委自然科学基金（CATC2009BB5063）;重庆市教委基金（KJ070317）

摘　　要：目的研究干扰素刺激基因ISG20对HCV的抑制作用及其机制，为探索丙型肝炎新的治疗方法提供依据。方法将含ISG20基因，其无功能突变体ISG20m和相应空白载体的质粒pcDNA3．1瞬时转染Huh7、Huh7．5和HEK293细胞，观察ISG20对HCV单顺反子复制子pSGRm-JFHlblaRL的抑制作用。同时将含ISG20、IsG20m基因和相应空白质粒的逆转录病毒载体pCX4Bsr转染Huh7．5细胞，建立稳定表达该基因的细胞系。将pSGRm-JFHlblaRL复制子转染ISG20细胞系，观察IsG20对该复制子的抑制作用。用细胞培养产生的HCV感染R粥20细胞系，探索其对HCV复制滴度的影响。数据比较用两因素多水平重复测量的方差分析。结果成功构建了稳定表达ISG20及其突变体的细胞系7．5-ISG20、7．5-ISG20m和质粒对照7．5-Bsr，免疫荧光证实ISG20和ISG20m表达于细胞质和细胞核。pSGRm-JFHlblaRLRNA在7．5-ISG20细胞中的复制被严重抑制，在所检测的各个时间点，其复制水平为7．5-ISG20m和7．5一Bsr细胞中的9．1％～16．7％。在Huh7、Huh7．5和HEK293细胞中，瞬时表达的ISG20对HCV复制子RNA也有明显的抑制作用，其复制水平为对照的16．7％～25．0％。HCV在7．5-ISG20细胞中扩增得到相似的结果，在感染后48～72h，HCV在7．5-ISG20细胞上清液中的滴度为7．5-ISG20m和7．5-Bsr细胞中的9．1％～12．5％。Westernblot实验结果显示，7．5-ISG20细胞中HCV杨心蛋白表达也明显减少。结论ISG20无论在瞬时转染的细胞和稳定表达的细胞系中均能抑制HCV复制子复制，并能减少HCV的扩增，这种抑制作用与ISG20的核酸外切酶作用密切相关。Objective To investigate the impact of exonuclease 20 kDa （ISG20） on replication of genotype 2a hepatitis C virus （HCV） subgenomic replicon RNA and infectivity of the cell culture-derived HCV strain JFH1 to determine the potential of exogenously expressed ISG20 as an anti-viral therapy of chronic hepatitis C. Methods Plasma vectors containing wild-type （WT） ISG20 or a catalyticallyinactive mutant ISG20m were transiently transfected into Huh7, Huh7.5 and HEK293 cells, and the replication of a monocistronic subgenomie JFH1 RNA replicon, SGRrn-JFH1BIaRL, was measured. Huh7.5 cells stably expressing ISG20, ISG20m, or the control vector were established by transducing replication incompetent pCX4- Bsr-myc relroviruses encoding WT ISG20, D94G mutant ISG20, or the crnpty vector, respectively, and selecting with 5 pg/mL of blasticidin for approximately three weeks. The stable Huh7.5 ceils were then transfected with HCV replicon RNA and infected with cell culture-derived HCV to investigate inhibition capacity of ISG20 against HCV. Results Huh7.5-ISG20, Huh7.S-ISG20m, and Huh7.5-Bsr controls cells stably expressing ISG20, ISG20m, or the control vector, respectively, were constructed successfully; the ectopically expressed ISG20 and ISG20m were distributed in both nucleus and cytoplasm, as detected by immunofluoreseence. SGRm-JFH1BlaRL replicated efficiently and with similar kinetics in the Huh7.5-Bsr and Huh7.5-ISG20m cells, with expression levels plateauing at 48-96 h post-transfection. In conWast, at all time points examined, SGRm- JFH1BIaRL replication was 9.1% to 16.7% in the Huh7.5-ISG20 cells. The Huh7, Hula7.5 and HEK293 cells transiently expressing ISG20 also showed 16.7% to 25.0% of HCV replication that the respective controls. In addition, the amount of infectious progeny JFI-II virus released in culture supemalants was 9.1% to 12.5% from the Huh7.5-ISG20 cells than from the Huh7.5-Bsr and HtthT.5-ISG20m cells at 48-72 h post-infection, and the latter two cultures produced similar JFH1 virus yields. F

关 键 词：干扰素 肝炎病毒 丙型 复制子 ISG20 体外研究 

分 类 号：R512.63[医药卫生—内科学]