HCV亚基因组复制子细胞培养上清外泌体HCV RNA的分离及其与胞内病毒水平的相关性  

HCV RNA levels in secreted exosomes correlate with intracellular viral replication levels in HCV subgenomic replicon cells

作　　者：王江华[1] 费然[1] 王雪艳[1] 张海莹[1] 魏来[1] WANG Jiang-hua FEI Ran WANG Xue-yan ZHANG Hai-ying WEI Lai(Peking University Peoplie＇s Hospital, Peking Uniresity Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, Beijing 100044, China)

机构地区：[1]北京大学人民医院北京大学肝病研究所丙型肝炎和肝病免疫治疗北京市重点实验室,北京100044

出　　处：《中国病毒病杂志》2016年第1期17-21,共5页Chinese Journal of Viral Diseases

基　　金：国家自然科学基金(81572043);国家科技重大专项(2012ZX10002003)

摘　　要：目的检测丙型肝炎病毒（HCV）复制子细胞培养上清中外泌体HCV RNA的稳定性和表达水平,并研究干扰素α抑制HCV复制过程中,外泌体HCV RNA水平与胞内病毒复制水平的相关性。方法建立G418筛选稳定表达的HCV复制子细胞模型HCV-Con I-Rep,比较超速离心和沉淀法对HCV RNA阳性外泌体的分离效果;实时定量PCR法检测培养上清的HCV RNA水平并评价其对表面活性剂NP-40和RNase A的敏感性;不同剂量干扰素α（0、0.4、2、10、50、250IU/ml）处理72h或100IU/ml干扰素α处理不同时间（0、6、12、24、48、72h）,分别检测上清和胞内HCV RNA水平,并评价两者的相关性。结果成功构建了稳定表达的HCV复制子细胞模型;PEG沉淀较超速离心法能够更高效地分离外泌体;HCV复制子细胞培养上清外泌体中含有较高水平（~106 IU/ml）的HCV RNA,但在表面活性剂的存在下,可因外泌体脂质膜破坏而被RNase A降解;干扰素α处理过程中,胞内HCV RNA和上清中外泌体HCV RNA水平均显著降低;Spearman相关性分析提示,胞内HCV RNA和上清中外泌体HCV RNA在不同剂量干扰素处理组和不同处理时间组的水平均具有显著相关性（r=0.9690,P〈0.01和r=0.7069,P=0.001）。结论 HCV亚基因组复制子细胞培养上清外泌体HCV RNA水平可作为更方便检测的指标,反映胞内病毒复制水平的变化。Objective To detect the HCV RNA level from exosome in the supernatant of HCV subgenomic replicon cells and study its correlation with intracellular HCV replication. Methods Huh7.5-based HCV replicon stable cell line was established by G418 screening from huh7.5cells transfected with neo gene containing HCV replicon RNA.Separation efficiency of HCV RNA positive exosomes were compared between ultracentrifugation and PEG precipitation methods.TaqMan real-time PCR was used to determine the intracellular HCV RNA levels and HCV RNA levels in exosome.Sensitivities of HCV RNA in exosome to RNase A were evaluated.Correlation of HCV RNA levels in exsomet with intracellular HCV RNA was evaluated in HCV replicon cells treated with interferon alpha. Results HCV replicon stable cell line expressing HCV subgenome was established.There were high amounts of HCV RNA packed within exosomes in the supernatant of replicon cells,which was resistant to RNase A treatment.Levels of HCV RNA from exosome and intracellular HCV RNA from HCV replicon cells were well correlated and could be inhibited by IFNαtreatment in dose-and time-dependent manners. Conclusions In HCV replicon cell model,levels of HCV RNA from exosome in the supernatant could reflect the intracellular viral replication rate.

关 键 词：丙型肝炎病毒 复制子 外泌体 干扰素Α 

分 类 号：R512.63[医药卫生—内科学]