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  • 罗正汉,刘燕,刘阳,唐海琳,徐铮昊,戚中田,赵平*.肝癌细胞乙酰胆碱酯酶表达水平与丙型肝炎病毒感染之间的相互影响[J].第二军医大学学报,2018,39(12):1302-1308    [点击复制]
  • LUO Zheng-han,LIU Yan,LIU Yang,TANG Hai-lin,XU Zheng-hao,QI Zhong-tian,ZHAO Ping*.Interaction between expression level of acetylcholinesterase and infection of hepatitis C virus in hepatoma carcinoma cells[J].Acad J Sec Mil Med Univ,2018,39(12):1302-1308   [点击复制]
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肝癌细胞乙酰胆碱酯酶表达水平与丙型肝炎病毒感染之间的相互影响
罗正汉,刘燕,刘阳,唐海琳,徐铮昊,戚中田,赵平*
0
(海军军医大学(第二军医大学)海军医学系生物医学防护教研室, 上海市医学生物防护重点实验室, 上海 200433
*通信作者)
摘要:
目的 探讨丙型肝炎病毒(HCV)感染对乙酰胆碱酯酶(AchE)表达水平的影响以及细胞内AchE活性的变化对HCV感染的影响。方法 以细胞培养来源的HCV(HCVcc)感染人肝癌细胞系Huh7细胞,用蛋白质印迹法以及实时荧光定量PCR(qPCR)进一步验证HCV感染对AchE表达水平的影响,AchE活性检测试剂盒检测细胞内AchE活性的变化。以AchE抑制剂多奈哌齐和伊托必利处理Huh7细胞,同时用HCVcc感染,通过蛋白质印迹法以及免疫荧光法检测Huh7细胞的HCV感染情况。用针对AchE基因的小干扰RNA(siRNA)转染Huh7细胞,随后用HCVcc感染Huh7细胞,通过蛋白质印迹法和免疫荧光法检测AchE的表达以及HCV感染情况。结果 HCVcc感染Huh7细胞60 h后,AchE蛋白表达水平升高,同时酶活性也增强(P均<0.01)。AchE抑制剂呈浓度依赖性抑制HCV对Huh7细胞的感染(P<0.01)。用siRNA敲低AchE的表达后,HCV对Huh7细胞的感染降低(P<0.01)。结论 HCV感染Huh7细胞能上调AchE的表达并增强AchE活性,AchE表达增加和活性增强都能促进HCV的感染,表明AchE在HCV感染Huh7细胞的过程中起着正反馈增强感染的作用。
关键词:  丙型肝炎病毒  黄病毒感染  乙酰胆碱酯酶  胆碱酯酶抑制剂  AchE活性
DOI:10.16781/j.0258-879x.2018.12.1302
投稿时间:2018-08-18修订日期:2018-10-09
基金项目:国家自然科学基金(31570170),国家科技重大专项(2017ZX10304403-6/7).
Interaction between expression level of acetylcholinesterase and infection of hepatitis C virus in hepatoma carcinoma cells
LUO Zheng-han,LIU Yan,LIU Yang,TANG Hai-lin,XU Zheng-hao,QI Zhong-tian,ZHAO Ping*
(Department of Biodefense, Shanghai Key Laboratory of Medical Biodefense, Faculty of Naval Medicine, Navy Medical University(Second Military Medical University), Shanghai 200433, China
*Corresponding author)
Abstract:
Objective To explore the effect of hepatitis C virus (HCV) infection on acetylcholinesterase (AchE) expression level and the effect of AchE activity on the infection of HCV. Methods Human hepatoma cell line Huh7 cells were infected with cell culture-derived HCV (HCVcc). AchE expression level was analyzed using Western blotting and real-time quantitative PCR (qPCR). AchE enzyme activity was assayed using AchE activity detection kit. Huh7 cells were treated with AchE inhibitor, donepezil or itopride, together with HCVcc infection, and then the HCV infection level of Huh7 cells was detected by Western blotting and immunofluorescence. Huh7 cells were transfected with small interfering RNA (siRNA) targeting to AchE gene, followed by infecting with HCVcc, and then the expression levels of AchE and HCV infection were analyzed by Western blotting and immunofluorescence. Results After 60 h of HCVcc infection, the expression level of AchE protein in Huh7 cells and the AchE activity were significantly increased (both P<0.01). AchE inhibitors significantly inhibited HCV infection of Huh7 cells in a concentration-dependent manner (P<0.01). After knocking down AchE with siRNA, HCV infection was significantly inhibited (P<0.01). Conclusion HCV infection of Huh7 cells can up-regulate the expression level of AchE and enhance the activity of AchE. The increase of AchE expression and activity can promote HCV infection, indicating that AchE plays a positive feedback enhancement effect on HCV infection of Huh7 cells.
Key words:  hepatitis C virus  flavivirus infections  acetylcholinesterase  cholinesterase inhibitors  AchE activity