稳定转染HBx基因的HepG2细胞对raf-1表达的影响  

Expression of raf-1 in HepG2 strain stably transfected by HBx gene

作　　者：曹鹏飞[1] 柳永和[1] 

机构地区：[1]中南大学湘雅医学院检验系,长沙410008

出　　处：《肿瘤研究与临床》2007年第11期724-726,共3页Cancer Research and Clinic

摘　　要：目的探讨乙型肝炎病毒 x（HBx）蛋白致癌变的分子机制。方法构建 HBx 基因真核表达载体 pCDNA3.1（+）-x,脂质体转染入 HepG2细胞（HepG2X 细胞）,以 pCDNA3.1空载体为对照（HepG2X₀细胞）,CA18选择培养,克隆扩增转染细胞,并用 Western blot 检测 X 蛋白的表达;MTS 实验检测细胞增生情况;流式细胞仪检测细胞周期变化;检测转染组及对照组 raf-1的表达变化。结果转染组 X 细胞在相对分子质量为21×10³位置有 X 蛋白的表达,而对照组 HepG2X₀细胞及未处理的 HepG2细胞未见 X 蛋白的表达。MTS 结果显示 X 细胞增生能力显著高于其他两株细胞（P【0.01）。流式细胞仪结果显示转染后的细胞系凋亡率增高,G₁/G₀期细胞减少,G₂期细胞增多。Western blot 结果示转染组 X细胞 raf-1表达明显较其他两组细胞增高。结论 HBx 有推进细胞周期的作用,使细胞快速进入 G₂期。其机制可能是 HBx 增加细胞周期蛋白 raf-1的表达。Objective To investigate the effect on cell cycle of hepatitis B virus x protein and ap- proach the molecular mechanism of x protein's carcinogenesis. Methods Gene transfection mediated by the lipofectamine was used to introduce the eukaryotic expression vector of HBV x gene into human hepato- cellular carcinoma cell line HepG2(HepG2X cell).The selective medium containing G418 was used to select the cell clones which the X protein was expressed constantly.Flow eytometry was used to detect the cycle of the cells;raf-1 protein was detected by Western blot.Results X protein could be expressed on 21×10~3 loca- tion in the transfected cells,while there were no protein expressed in the control cells.The proliferation of X cells increased significantly according to MTS method(P<0.01).The results of flow cytometry also manifested the number of HepG2 cycle phase cells was also more than the control cell lines. The result of Western bolt discovered that the expression of raf-1 protein increased in the transfccted cells.Conclusion The X protein can stimulate the growth of hepatoeellular carcinoma cells.The mechanism maybe HBx gene can enhance the expression of raf-1.

关 键 词：肝炎病毒 乙型 X 蛋白 细胞周期 raf-1蛋白 

分 类 号：R735.7[医药卫生—肿瘤]