乙型肝炎病毒X基因转染人肝癌细胞株双向凝胶电泳图谱分析

目的 研究转染HBV X基因的人肝癌细胞株中蛋白质表达谱的改变,为筛查在HBV相关性肝细胞癌发生中发挥重要作用的关键蛋白质分子奠定基础.方法 利用分子生物学方法建立稳定表达HBV X蛋白(HBx)的肝癌细胞株HepG2+HBx,同时设空载体peDNA3转染细胞HepG2-pcDNA3及HBV全基因转染的人肝癌细胞株HepG2.2.15为对照.PCR法扩增Neo基因检测质粒DNA片段的插入,免疫印迹法检测HBx蛋白的表达.利用固相pH梯度双向凝胶电泳分离3种肝癌细胞株HepG2-pcDNA3、HepG2-HBx和HepG2.2.15的总蛋白,用图像分析软件比较、分析、识别细胞间的差异表达蛋白质.统计学分析采用t检验.结果 获得分辨率高、重复性好的3种细胞的双向电泳(2-DE)图谱.软件分析表明,HepG2-pcDNA3、HepG2-HBx和HepG2.2.15细胞的2-DE凝胶可识别蛋白点分别为(2 095±137)、(2 188±105)和(2 109±20)个.比较HepG2-pcDNA3与HepG2-HBx细胞的2-DE图谱发现37个差异显著的蛋白点,其中21个在HepG2-HBx表达上调,16个下调.6个表达量差异在5倍以上(t=0.027,P＜0.05);HepG2.2.15与HepG2-HBx细胞相比,有38个差异显著的蛋白点,其中35个在HepG2-HBx细胞中上调,3个下调,14个表达量差异在5倍以上(t=0.031,P＜0.05).结论 HBx基因转染引起人肝癌细胞株蛋白质表达谱的变化,可能与感染的肝细胞发生恶性转化的分子生物学机制有关.

Two-dimensional gel eleetrophoresis analysis of differentially expressed proteins in human hepatocellular carcinoma cell line HepG2 transfected with hepatitis B virus X gene

Objeetive To study protein expression profiles in human hepatocellular carcinoma (HCC)cell line HepG2 transfeeted with hepatitis B virus X gene(HBX),and to provide information for identification of key proteins in hepatitis B virus(HBV)related HCC.Methods HepG2-HBx cell strains stably expressing HBV X protein(HBx)were constructed using molecular biological methods.HepG2-pcDNA3 cells which were constructed with HepG2 cells were transfeeted with plasmid pcDNA3 and HepG2.2.15 cells transfected with HBV whole genome were used as controls.The integration of the exogenous vector DNA was detected by Neo gene polymerase chain reaction(PCR).The HBx expression was deteeted bv Western blot.Total cellular proteins were extracted from HepG2-peDNA3,HepG2-HBx and HepG2.2.15 cells and separated by immobilized pH gradients twodimensional gel electrophoresis(2-DE).The differential protein-spots between HepG2-pcDNA3,HepG2-HBx and HepG2.2.15 cells were identified using image analysis software.The statistical analyses were done by t test.Results The well-resolved,reproducible 2-DE patterns of HepG2-peDNA3,HepG2 HBx and HepG2.2.1 5 cells were obtained.The average protein-spots of HepG2-peDNA3,HepG2-HBx and HepG2.2.15 cells identified by 2 DE were 2 095±137,2 188±105 and 2 109±20,respectively.There were 37 differential protein spots between HepG2-HBx and HepG2-peDNA3 cells,of which 21 were up-regulated and 16 were down-regulated in HepG2-HBx cells.Expressions of 6 proteins were 5 times differences between HepG2-HBx and HepG2-pcDNA3 cells (f=0.027,P＜0.05).Compared with HepG2.2.15 cells,there were 38 differential protein spots,of which 35 were up-regulated while 3 were down-regulated in HepG2-HBx cells.Expressions of 14 proteins were 5 times differences between HepG2-HBx and HepG2.2.15 cells(t=0.031,P＜0.05).Conclusion HBx gene transfection results in changes of protein expressions of human HCC celIlines,which might be one of the molecular mechanisms of hepatocarcinogenesis in HBV infected hepatocytes.