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

发布时间：2018-04-17 05:26

本文选题：丙型肝炎病毒 + 复制子　；参考：《中国病毒病杂志》2016年01期

【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,P0.01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。
[Abstract]:Objective to detect the stability and expression level of HCV RNA in the culture of hepatitis C virus (HCV) replicator cells, and to study the correlation between the HCV RNA level of exosome and the replication level of intracellular virus during the inhibition of HCV replication by interferon 伪.Methods G418 was established to screen stable HCV replicator cell model HCV-Con I-Rep. to compare the effect of ultracentrifugation and precipitation on the isolation of HCV RNA positive exosomes.The level of HCV RNA in culture supernatant was measured by real-time PCR and its sensitivity to surfactant NP-40 and RNase A was evaluated.The correlation between them was evaluated.Results the stable expression of HCV replicator cell model was successfully constructed. Compared with ultracentrifugation, HCV precipitation was more efficient in the isolation of HCV RNAs from the exocrine of the exocrine cell culture supernatant, but in the presence of surfactant.During the treatment of interferon 伪, the levels of HCV RNA and HCV RNA in the supernatant decreased significantly.The levels of intracellular HCV RNA and supernatant HCV RNA were significantly correlated with those of different doses of interferon treatment group and different treatment time groups (P0.01 and 0.7069 P0. 01).Conclusion the level of HCV RNA in the supernatant of HCV subgenomic replicator cell culture can be used as a more convenient index for detection and reflect the changes of the replication level of intracellular virus.
【作者单位】：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;
【基金】：国家自然科学基金(81572043) 国家科技重大专项(2012ZX10002003)
【分类号】：R512.63

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