丙型肝炎病毒E1蛋白活化MAPK／ERK信号通路促进肝癌细胞增殖  

Hepatitis C Virus E1 Protein Promotes Hepatoma Cell Growth Through MAPK/Erk Signaling Pathway

作　　者：陈庆美[1] 刘娇[1] 单晓亮[1] 周帆[1] 权会琴[1] 陈林林[1] 唐霓[1] 

机构地区：[1]重庆医科大学附属第二医院病毒性肝炎研究所感染性疾病分子生物学教育部重点实验室,重庆市400016

出　　处：《医学分子生物学杂志》2012年第1期42-48,共7页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.30972586,31171307）;再庆市自然科学基金重点项目（No.CSTC2009BA5036）

摘　　要：目的研究丙型肝炎病毒（hepatitisCvirus，HCV）编码蛋白E1的生物学功能。方法分别构建编码HCV重要蛋白E1、E2、NS3、NS5a、NS5b的腺病毒载体Ad—E1、Ad—E2、Ad—NS3、Ad—NS5a、Ad—NS5b；将重组并包装的腺病毒分别感染SMMC-7721细胞，测定感染滴度，通过RT—PCR方法在转录水平鉴定HCVE1、E2、NS3、NS5a、NSSb的表达，用Western印迹在蛋白水平鉴定E1蛋白的表达。腺病毒感染SMMC-7721细胞后，通过细胞增殖实验筛选生物学功能最明显的蛋白。将筛选到的Ad—E1感染SMMC-7721细胞，用MTS、结晶紫、细胞周期实验观察体外过表达E1蛋白对感染细胞增殖的影响；Western印迹检测p-ERK、ERK的表达；RT—PCR检测c—Myc、cyclinD1、c—Jun、c．Fos基因的表达。结果成功扩增了能够编码HCV重要蛋白E1、E2、NS3、NS5a、NS5b的高滴度腺病毒Ad—E1、Ad—E2、Ad—NS3、Ad—NS5a、Ad—NS5b，并且通过RT—PCR方法在转录水平鉴定了目的基因的表达，Western印迹方法在蛋白水平鉴定了E1蚩白的表达。通过细胞计数、MTS、结晶紫实验证实Ad—E1感染组细胞较对照组增殖速度加快，细胞周期显示Ad-E1感染组细胞34．38％处于S期，明显高于Ad—GFP（27．32％）（P〈0．05）对照组；Ad-E1感染绢p-ERK蛋白表达量增高，同时与细胞增殖相关的MAPK／ERK下游基因转录水平上凋。结论体外过表达HCVE1蛋白可以明显促进SMMC-7721细胞的增殖，其促增殖作用可能与MAPK／ERK信号通路的活化相关。Objective To investigate the biological function of E1 protein encoded by Hepatitis C virus （HCV） . Methods SMMC-7721 cells were infected with adenovirus Ad-E1, Ad-E2, Ad-NS3, Ad-NSSa and Ad-NSSb, which encodes El, E2, NS3, NSSa and NSSb protein, re- spectively. Over-expression of the target gene was identified by Western blot or RT-PCR meth- ods. Cells were infected with high titer of adenovirus. Biological function induced by different viral protein was determined by cell proliferation assays. The cell growth and cell cycle were analyzed by MTS, Crystal Violet assay and flow cytometry. The transcriptional factor and target genes of MAPK/ ERK signal pathway were determined by Western blot or RT-PCR methods. Results RNA transcript products of El, E2, NS3, NS5a and NS5b were identified by RT-PCR method respectively. El Protein expression was verified by Western blot analysis. Cell growth curve, MTS and cell viability assay indicated that over-expression of HCV E1 alone resulted in an increased proliferation rate in SMMC-7721 cells. Cell cycle analysis showed that the S-phase distribution in cells transduced with E1 was 34.38 %, much higher than that of GFP control （27.32 % ） （P 〈0. 05） . Moreover, E1 moderately up-regulated mRNA expression levels of several transcriptional factors downstream of MAPK/ERK signal pathway. Conclusion The growth effect of overexpressed HCV E1 Protein in vitro on SMMC-7721 cells was partially correlated with MAPK/ERK signal pathway.

关 键 词：丙型肝炎病毒 E1蛋白 丝裂原活化蛋白激酶 细胞增殖 

分 类 号：R512.63[医药卫生—内科学]