论文部分内容阅读
目的探讨HBx蛋白对人肝细胞发生侵袭恶性表型的影响和作用机制。方法利用固相pH梯度双向凝胶电泳分离稳定表达HBx基因的CCL13-HBx细胞系及转染空质粒的CCL13-pcDNA3.1细胞系的总蛋白质,凝胶经蓝银显色后,采用Image Master 2-DE Elite 4.01图像分析软件进行比较分析、识别差异表达的蛋白质,MALDI-TOF-MS质谱仪得到相应的肽质指纹图谱后,搜索数据库鉴定部分差异蛋白质点。结果获得了背景清晰、分辨率高、重复性好的CCL13-HBx和CCL13-pcDNA3.1细胞总蛋白,图像分析识别差异蛋白质点共26个,通过质谱分析鉴定出14个非冗余的与细胞代谢、细胞周期及信号转导相关的差异表达蛋白质。结论建立了CCL13-HBx,CCL13-pcDNA3.1细胞系的2-DE图谱,鉴定了14个HBx诱发肝癌发生的相关蛋白质,为在蛋白质水平阐明HBx的诱发肝癌发生的机制提供了新线索。
Objective To investigate the effect of HBx on malignant phenotype of human hepatocytes and its mechanism. Methods The total protein of CCL13-HBx cell line stably expressing HBx gene and CCL13-pcDNA3.1 cell line transfected with empty plasmid was separated by two-dimensional gel electrophoresis using solid-phase pH gradient. The gel was stained with blue silver and then analyzed by Image Master 2-DE Elite 4.01 image analysis software for comparative analysis to identify differentially expressed proteins. After the corresponding peptide fingerprinting was obtained by MALDI-TOF-MS mass spectrometry, some differential protein spots were identified by searching databases. Results The total proteins of CCL13-HBx and CCL13-pcDNA3.1 cells with clear background, high resolution and good reproducibility were obtained. Twenty-six protein spots were identified by image analysis and 14 non-redundant cells were identified by mass spectrometry Metabolism, cell cycle and signal transduction related to the differential expression of proteins. Conclusions The 2-DE map of CCL13-HBx and CCL13-pcDNA3.1 cell lines was established and 14 HBx-induced hepatocellular carcinomas were identified, providing new clues for elucidating the mechanism of HBx-induced hepatocellular carcinoma at the protein level.