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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 stability and expression level of exosome HCV RNA in the culture supernatant of hepatitis C virus (HCV) replicon cells and to study the effect of interferon α on inhibiting the HCV RNA replication and the level of intracellular viral RNA Relevance. Methods The HCV replicon cell model HCV-Con I-Rep was screened by G418. The isolation of HCV RNA positive exosomes by ultracentrifugation and precipitation was compared. Real-time quantitative PCR was used to detect the level of HCV RNA in the culture supernatants. Its sensitivity to surfactant NP-40 and RNase A; different doses of interferon alpha (0,0.4,2,10,50,250 IU / ml) for 72h or 100IU / ml interferon alpha at different times (0, 6, 12, 24, 48, 72h). The levels of HCV RNA in the supernatant and intracellular were detected, and the correlation between them was evaluated. Results The HCV replicon cell model was established successfully. PEG pellets were isolated more efficiently by ultracentrifugation than exosomes. HCV replicon cell culture supernatants contained higher levels of exosomes (-106 IU / ml) Of HCV RNA, but was degraded by RNase A in the presence of surfactants due to disruption of the exosome lipid membrane; both intracellular HCV RNA and exosome HCV RNA levels in the interferon alpha treatment were significant Spearman correlation analysis showed that there was a significant correlation between intracellular HCV RNA and exosome HCV RNA in different doses of interferon treatment group and different treatment time groups (r = 0.9690, P <0.01 and r = 0.7069, P = 0.001). Conclusion The HCV RNA level in exosomes of HCV subgenomic replicon cell culture can be used as a more convenient indicator to detect the intracellular viral replication.