【作者】 郭丹华；

【导师】 林旭； 林建银；

【作者基本信息】 福建医科大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 乙型肝炎病毒基因组剪接变异体( spliced variants of hepatitis B virus genomes)是一大类基因部分缺失的亚基因组DNA,由乙型肝炎病毒(Hepatitis B virus, HBV)前基因组RNA (pregenomic RNA, pgRNA)经剪接并逆转录产生[1]。在已经分离到的HBV剪接变异体中,长度约为2.2kb的剪接变异体占80%[2],此类变异体主要分为单剪接与双剪接两种类型。研究显示这两种HBV基因组剪接变异体编码的剪接特异性新蛋白与病毒的持续性感染及致病性相关[3,4],但其确切的致病机制迄今不明。本研究通过人类基因表达谱芯片研究分析这两种剪接特异性新蛋白对Huh7肝细胞基因表达的影响,探寻它们可能的致病机制。本研究第一部分旨在构建剪接特异性新蛋白真核表达载体,并证实其在肝细胞中的表达。为此将剪接特异性新蛋白编码区克隆到真核表达载体pcDNA3.1/HisC ,获得重组载体pcDNA3.1/HisC-TPds(双剪接型)、pcDNA3.1/HisC-TPss(单剪接型),瞬时转染Huh7肝细胞,以Anti-Xpress抗体进行Western Blot检测融合蛋白,证实剪接特异性新蛋白可在Huh7肝细胞中表达。本研究第二部分以pcDNA3.1/HisC、pcDNA3.1/HisC-TPds、pcDNA3.1/HisC-TPss分别瞬时转染Huh7肝细胞,提取转染细胞总RNA,进行人类基因表达谱芯片分析。同时以半定量RT-PCR验证芯片结果。芯片分析显示:2.2kb HBV基因组剪接变异体剪接特异性新蛋白可能从干扰肝细胞骨架的重塑、细胞内物质代谢等方面引起肝细胞的增生、迁移及代谢异常,促进肿瘤的发生。更多还原

【Abstract】 Spliced variants of hepatitis B virus genomes are a group of subgenomic length DNA generated from 3.5Kb pregenomic RNA by splicing and reverse transcription. More than 80% of these spliced variants are 2.2Kb doubly or singly spliced variants of hepatitis B virus genome, which could encode splicing-specific novel proteins and closely related with persistent infection and pathogenicity of HBV with unclear mechanism. Inorder to explore the potential pathogenicity of the novel proteins encoded by the 2.2Kb doubly or singly spliced variants of HBV genome, we investigated the effection of the novel proteins on Huh7 hepatocytes by gene expression profile array assay.The first part of this study was aimed to construct the splicing-specific proteins recombinant eukaryotic expression vectors and confirm their expression in Huh7 hepatocytes. To address this issue, the splicing-specific coding regions were cloned separately into the eukaryotic expression vector pcDNA3.1/HisC to construct the recombinant eukaryotic expression vectors pcDNA3.1/HisC-TPds(harboring the double spliced splicing-specific novel protein) and pcDNA3.1/HisC-TPss (harboring the signle spliced splicing-specific novel protein). The recombinant plasmids were transiently transfected into Hhu7 hepatocytes separately, and Western Blot using anti-Xpress antibody was used to confirm the expression of TPds and TPss.In the second part of this study , total RNA of cells transiently transfected with pcDNA3.1/HisC , pcDNA3.1/HisC-TPds and pcDNA3.1/HisC-TPss were extracted respectively and used for gene expression profile array assay. Semi-quantitative RT-PCR were introduced to validate the array assay. The result demonstrated that the splicing-specific novel proteins encoded by the 2.2Kb spliced variants of HBV genome may disturb the re-orgnazation of the cystoskeleton and the metabolism of Huh7 hepatocytes , which result in the abnormality of cell proliferation , mobility and metabolism, and eventually attributed to the carcinogenesis of Hepatocellular Carcinoma.更多还原