【作者】 徐浩；

【导师】 赵龙凤； 成军；

【作者基本信息】 山西医科大学 ， 传染病学， 2008， 硕士

【摘要】 乙型肝炎病毒(hepatitis B virus,HBV)是慢性肝炎的重要病原之一,常导致严重肝病,包括肝硬化和肝细胞癌(hepatocellular carcinoma,HCC)。在HCC形成过程中,其中HBxAg(hepatitis B x antigen)的功能一直是研究热点。在转录水平HBxAg对病毒本身及人类多种基因具有激活及阻遏作用。S100A11蛋白广泛存在于正常人体组织细胞中,该蛋白进入细胞核发挥细胞生长负调节作用。在某些肿瘤细胞中S100A11蛋白的表达量升高[12-14]。而增多的S100A11蛋白从肿瘤细胞的细胞核移位至细胞质。“永生化”细胞中的这种蛋白定位改变可能使其丧失本身的生物学功能。目的本课题旨在研究S100A11蛋白在肝癌细胞系BEL-7402增殖过程中发挥的作用,以及明确该蛋白在正常肝细胞、慢性HBV感染的肝细胞及肝癌细胞中的核质定位分布特点,进一步探讨HBxAg对S100A11基因在转录水平的调节作用。方法1.构建S100A11的真核表达载体(实验组),同时设平行对照,转染肝癌细胞系BEL-7402,筛选稳定表达。通过流式细胞术检测肝癌细胞BEL-7402中处于细胞分裂各周期中的相对细胞数,分析含目的基因质粒组及相应对照组中处于分裂周期中S期的细胞数。采用活细胞发光法检测两组细胞中活细胞数量相对值,绘制细胞生长曲线并进行比较。2.构建HBxAg的真核表达载体及S100A11-promoter荧光素报告质粒,并设平行对照,转染BEL-7402。制备转染后的细胞裂解液,在肝癌BEL-7402细胞中检测HBxAg对S100A11启动子转录活性的影响。用Luciferase法检测报告基因表达。3.通过免疫组织化学技术比较S100A11蛋白在正常肝细胞、慢性HBV感染的肝细胞以及肝癌细胞中的分布情况。构建S100A11绿色荧光素质粒,瞬时转染肝癌细胞系BEL-7402,通过荧光显微镜观察融合蛋白在BEL-7402中的分布。结果1.实验组BEL-7402细胞经流式细胞术检测后发现处于S期的细胞占26.49±1.94%;而平行对照组中处于S期的细胞占30.42±1.62%;野生株BEL-7402中处于细胞分裂周期中S期的细胞占总细胞数的31.40±2.06%。均分细胞后,用活细胞发光法检测细胞增殖情况。结果显示,36 h检测时实验组细胞增殖速率与对照组相比明显降低。2.本实验成功获得含人S100A11基因启动子的荧光素报告载体,且测序证实插入序列与理论序列一致。实验组的转录活性与对照组的转录活性相比降低60%。3.免疫组织化学染色发现,肝癌细胞胞质中S100A11蛋白的量较正常细胞明显增多,慢性乙型肝炎中S100A11蛋白的表达量微弱。而我们强制表达的外源性S100A11蛋白在BEL-7402细胞的细胞核及细胞质中均有分布。结论1.S100A11对肝癌细胞BEL-7402具有明显的生长抑制作用,主要表现为使处于细胞分裂周期中S期细胞数量明显减少。2.成功获得人S100A11基因启动子序列,HBxAg对S100A11基因的转录表达具有抑制作用。提示HBV通过HBxAg可使S100A11蛋白的表达下调,这可能使经该途径发挥的细胞生长抑制作用减弱。3.在HCC的发生发展中,可能存在S100A11蛋白的核质移位现象。慢性乙型肝炎细胞中S100A11蛋白的表达微弱,过表达的S100A11蛋白在BEL-7402细胞中为核质泛表达。更多还原

【Abstract】 Hepatitis B virus (HBV) is the major cause of chronic hepatitis, It often leads to severe liver diseases, including liver cirrhosis and hepatocellular carcinoma (HCC). In the course of HBV related HCC development, the function of HBxAg has been paid close attention recently[1-4]. HBxAg has transcriptional activation as well as transcriptional inhibition[5-8]. S100A11 gene is exsibed in most of human tissue cells, it moves to nucleus and plays a negative regulation on cell growth [9-11]. Investigators discover that the expression of S100A11 protein is increased in some cancer cell[12-14]. Further investigation discovered that increased expression protein S100A11 is transferred from nucleu to cytoplasm in cancer cell[15], which probably makes it lose it’s virgin biological function.Objective This study aimed to uncover the function that S100A11 protein playing on BEL-7402 cell growth and identify it is disposition feature in normal hepatic cell, chronic HBV infected hepatic cell and HCC further investigate the effect of transcriptional regulation of HBx on S100A11 gene.Methods1. The expression vector S100A11 (experimental group) was constructed and transfected BEL-7402 cells. Stabilized expression was screened. The rate in BEL-7402 S phase is detected by FCM. Living cell rate of two groups were detected by Celltiter-Glo luminescent cell viability assay.2. HBx recombined expression plasmid and S100A11-p promoter reporters vector were constructed, meanwhile set up paralleled control, Transfected into BEL-7402 cell. After transfection, cell lysate was prepared. Detection the effect of transcription activity that X gene on S100A11 promotor in BEL-7402. reporter gene expression was detect by Luciferase.3. Immunohistochemistry (IHC) was used to compare S100A11 location in normal hepatic cell, chronic HBV infected hepatic cell and HCC. Recombinant green fluorescence plasmid of S100A11 was transiently transfected BEL-7402. Fusion protein in BEL-7402 was observed by fluorescence microscope.Results1. In control group and mocked transfected group, BEL-7402 cells were detected by FCM and S phase cell population in each group was 26.49±1.94% and 30.42±1.62% respectively.Celltiter-Glo luminescent cell viability assay showed that after 36 hour of transfection, the proliferation was obviously decreased in research group than control.2. Successfully, the gene inserted into the recombined vector was sequenced and compared with that of GenBank. Compared with the first group which cotransfected with pGL4.10-S100A11-promoter and pcDNA3.1(-)-HBX the promoter activity of the second group which cotransfected with pcDNA3.1(-) and pGL4.10-S100A11-promoter was obviously decreased. fluorescein reporters is 1/3 fold lower.3. The result showed that S100A11 is expressed mostly in cytoplasm in HCC than normal hepatic cell. S100A11 is obviously decreased in chronic HBV infected hepatic cell. The transfected exogenous S100A11 was expressed both in nucleus and cytoplasm of BEL-7402.Conclusions1. anti-oncogene S100A11 has obvious growth inhibiting effect on BEL-7402.2. The correct sequence of S100A11gene promoter is successfully obtained, HBxAg has inhibiting effect on transcription and expression of S100A11 gene. indicating that HBV may play carcinogenesis role by regulating anti-oncogene effect of x protein.3. S100A11 is possibly immgrated from nucleus to cytoplasm in HCC happen and discoverment. S100A11 is weakly expressed in cytoplasm. The higher expressed S100A11 could be seen in both nucleus and cytoplasm of BEL-7402.更多还原