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目的采用核素标记相对和绝对定量(isobaric tags for relative and absolute quantitation,i TRAQ)蛋白组学技术发现并鉴定血源性丙型肝炎病毒(blood borne hepatitis C virus,bb HCV)感染人胚胎肝干细胞(human fetal liver stem cells,h FLSCs)的新关键因子。方法按照bb HCV感染与否以及感染后不同时间点将细胞分为5组,去除各组细胞裂解后的高丰度蛋白,运用纳升液相色谱仪(LC-ESI-MSMS)发现并鉴定差异表达蛋白,并对差异表达蛋白进行生物信息学分析,最后对所鉴定出的蛋白利用RT-q PCR和Western blot进行验证。结果对各个时间点的对照组和感染组间进行差异蛋白富集分析,得到bb HCV感染h FLSCs过程中表达差异蛋白PTPRA。针对鉴定的蛋白PTPRA,利用RT-q PCR和Western blot检测发现bb HCV感染后其表达明显升高;利用小RNA技术干涉PTPRA可以降低病毒进入细胞的量。结论 PTPRA可能在bb HCV感染侵入h FLSCs和子代病毒分泌过程中发挥着重要的作用。
OBJECTIVE: To detect and identify human borne hepatitis C virus (bb HCV) infected human embryonic liver stem cells by using isobaric tags for relative and absolute quantitation (i TRAQ) proteomics. (human fetal liver stem cells, h FLSCs) of the new key factor. Methods The cells were divided into 5 groups according to the infection of bb or not and the different time point after infection, and the high abundance protein of each group was removed. LC-ESI-MSMS identified and identified the difference The proteins were expressed and analyzed by bioinformatics. Finally, the identified proteins were verified by RT-q PCR and Western blot. Results The differential protein enrichment analysis was performed between the control group and the infected group at each time point, and the differential expression protein PTPRA was obtained during the h FLSCs infection by bb HCV. According to the identified protein PTPRA, RT-q PCR and Western blot showed that the expression of PTPRA was significantly increased after infection with bb HCV, and the interference of PTPRA with small RNA could reduce the virus entry into the cell. Conclusions PTPRA may play an important role in the invasion of h FLSCs by bb HCV infection and the secretion of progeny virus.