【作者】 丁浩；

【导师】 冯涛；

【作者基本信息】 重庆医科大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 目的:利用HBX腺病毒质粒载体,转染人肝细胞株L02,观察HBX对?-catenin表达量及转录活性的影响,研究HBX与Wnt/β-catenin信号转导通路中主要信号分子的相互作用,确定HBX与β-catenin的内在联系,旨在探讨HBV相关性HCC的发生机制,阐明Wnt/β-catenin信号转导途径在胚胎发育和肿瘤发生过程中的重要作用。方法:构建HBX的腺病毒(Adenovirus)质粒,转染肝细胞株L02,确定HBX在肝细胞中高效表达;Western blotting检测转染后细胞中?-catenin表达水平的改变;荧光素酶报告基因检测?-catenin的活性变化;免疫共沉淀分析HBX与GSK3β、Axin的相互作用,初步探讨HBX对Wnt/β-catenin信号转导通路发生影响的作用机理。结果:HBX在肝细胞中的表达:重组腺病毒质粒能有效转染L02细胞,转染效率较高,当MOI为80时,细胞感染效率为70%左右;并通过western blotting在转染Ad-HBX-GFP的L02细胞中检测到HBX的稳定表达。免疫印迹法检测结果:转染Ad-HBX-GFP组和转染Ad-GFP组以及未转染腺病毒载体组(空白组)三组蛋白在相对分子量93KD附近检测到一条显色区带,转染Ad-GFP组和空白组的条带更多还原

【Abstract】 Objects: To investigate the interaction between HBX protein and beta-catenin, illuminate the molecular mechanism in HBV-associated hepatocellular carcinoma (HCC) carcinogenesis and the important role of wnt/β-catenin signaling pathway in the generation/differentiation of many tissues.Methods: Western blotting analysis and Luciferase Assay methods were used to detect the expression and transcriptional activity of beta-catenin in normal liver cell line (L02) infected by adenoviruses with HBX gene. Co-Immunoprecipitation was used to investigate the interaction between HBX proteins and the main signal molecular of Wnt/β-catenin signaling pathway.Results: Western blotting: the control group and Ad-GFP infected group expressed low beta-catenin protein, 102.18±2.56 and 101.45±3.66 respectively, P>0.05, there was no significance between groups; however the expression of beta-catenin protein was greatly enhanced in Ad-HBX-GFP infected group (132.11±12.48), P<0.05, there was significance between groups. Luciferase Assay: infection of HBX更多还原