【作者】 张昊；

【导师】 王瑞林； 樊青霞；

【作者基本信息】 郑州大学 ， 肿瘤学， 2003， 硕士

【摘要】 一、背景 肝细胞肝癌(Hepatocellular Carcinoma，HCC)是我国较为常见的一种恶性肿瘤，在我国的肿瘤发病率中排行第三。肝癌的病因迄今尚未完全阐明，既往的许多研究表明，肝癌的发生与环境因素有密切关系，其中乙型肝炎病毒感染、不良生活方式(如吸烟和饮酒)以及暴露于环境致癌物(如黄曲霉毒素)等是导致肝癌的重要原因。但即使在肝癌高发区，接触同样的环境因素仅有少数人发病，乙肝病毒阳性病人中也只有一小部分人发展成肝癌；提示在相同的暴露条件下，个人的易感性因素可能是促使发病的关键。然而什么是中国人肝癌的个体易感性因素目前尚不十分清楚。通过对乙肝病毒导致肝癌的发病机理的研究发现，乙肝病毒基因组中的蛋白HBx是引起肝细胞肝癌的重要蛋白，该蛋白可与细胞中的癌症相关的多种蛋白相互作用；最近人们发现HBx可以与DNA损伤结合蛋白DDB1作用，从而抑制该蛋白与DNA的结合。DDB1是DNA修复系统中核苷酸切除修复途径的重要蛋白，HBx蛋白与DDB1的结合可以降低细胞的核苷酸切除修复能力。对肝细胞肝癌的EST分析也发现DNA修复相关基因DDB2、RAD51C、RAD23A、ERCC1和LIG1表达的下调。在过量表达DNA修复基因MGMT的转基因小鼠中也发现自发性肝细胞肝癌发病率降低。这些提示乙肝病毒在肝细胞癌变过程中，可能是通过影响DNA损伤修复来起作用的，DNA修复基因在肝细胞肝癌的发生过程中起着重要作用。 DNA损伤普遍存在，而细胞也已进化产生复杂的DNA修复体系来修复损伤，保持基因组的完整性。人体重要的DNA修复系统主要有四种，碱基切除修复为其中的一种，其修复过程首先由DAN糖苷酶切除受损伤碱基，形成AP位点；然后由AP裂解酶切开，单链断裂的保护，DNA聚合酶补齐，连接酶连接。hOGG1基因和MBD4基因即为DNA糖苷郑州大学硕士论文DNA修复基因单核昔酸多态与肝细胞肝癌遗传易感性酶基因。本课题应用基因组技术，通过对hoGGI基因和MBD4基因的两个高频单核普酸多态的研究，了解它们与肝癌之间的关系，从而更深入了解肝癌的发病机制，为肝癌预防、诊断和治疗提供理论依据。 二、材料与方法 1、基本资料 本课题采用病例一对照研究，包括96例肝细胞肝癌患者和96例正常对照，病例和对照均来自河南省，所有病例经临床资料和组织病理学检查确诊为肝细胞肝癌，所有研究对象没有直系血缘关系，同时收集每个研究对象的详细人口学资料，吸烟、饮酒和乙肝病毒感染情况。抽取病例和对照的外周血5毫升，加构栋酸钠抗凝，一20“c冻存。 2、方法 提取血样DNA，应用聚合酶链反应(po 1 ymerase Chain reactionPCR)技术，对hOGGI基因多态位点Ser326CyS和MBD4基因的多态位点Glu346LyS进行基因扩增，用凝胶电泳证实待检测片段存在，然后纯化，最后经基因测序，检测h0GGI基因的遗传变异ser326cys和MBD4基因的遗传变异G 1 u346LyS在肝细胞肝癌患者和正常对照中等位基因和基因型的变化，运用Pol yphred软件，对测序结果进行分析，通过统计分析了解它们与肝细胞肝癌风险的关系。 技术路线外周血尽提取DNA尽PCR扩增尽3%凝胶电泳郑州大学硕士论文DNA修复基因单核昔酸多态与肝细胞肝癌遗传易感性尽纯化尽 测序 3、统计分析 以比值比(odds RatioS，ORS)及其95%置信区间(Confideneeinterva1s，c工s)表示相对风险度。所有的统计检验均为双侧概率检验。所用统计软件为statist1CAnalysissystem第6版(SASInsititute，Cary，NC，USA)。 三、结果 不同个体的hOGGI基因第7外显子有三种基因型分别为Cys/eys、eys/ser和 ser/ser。hoGG一基因的等位基因Cys基因频率在肝细胞肝癌病人中要高于正常对照(病例43%，对照31%)，肝癌病例组的Cys/Cys、CyS/Ser、Ser/Ser的基因型频率分别为20.9%、44.2%和34.9%，基因型Cys/CyS和Cys/Ser与Ser/Ser相比在病人中的频率都要高于正常对照，表现出剂量效应.其中携带Cys/Cys基因型的个体患病风险增加约2倍(OR=1.9)。正常人群cys等位基因频率为33.1%，肝癌病例组为43%，肝癌病人要明显高于正常对照。 不同个体MBD4基因第3外显子有e lu/Glu、Glu/Lys、Lys/Lys三种基因型。正常人群Glu/Glu、Glu/LyS、LyS/LyS基因型频率分别为40.4%、46.8%和12.8%。肝癌病例组为41.7%、48.8%和9.5%。等位基因Lys频率在病例和对照分别为33.9%和36.2%，两组比较无显著性差异。 四、结论1、h0GGI基因的等位基因CyS可能增加肝细胞肝癌的患病风险其第7外显子存在ser326CyS多态，可能是中国人肝癌的易感性多态。 2、MBD4基因的遗传变异G 1 u346LyS与肝细胞肝癌的发生无明郑州大学硕士论文DNA修复基因单核昔酸多态与肝细胞肝癌遗传易感性显相关性。3、如果本研究结果得到进一步证实，则通过检测外周血DNAhoGGI基因ser326cys多态，可以为临床早期诊断肝癌提供重要参考并可以筛查肝癌的高危人群。更多还原

【Abstract】 BackgroundHepatocellular carcinoma (HCC) rank third in frequency in China. The pathogenesis of HCC is atill under discussion. Many reports show that the pathogenesis of HCC was strong associated with environmental factor, including hepatitis B virus (HBV) infection, alcohol consumption, smoking and carcinogens exposure (aflatoxin, for example). But only a few people developed HCC even though they live in the geographic areas at highest risk of HCC or they are HBV infected. This suggests that the individual susceptibility plays a in portent role in the pathogenesis of HCC. The susceptible genetic marker of HCC is not clear. We know that the NDA of HBV codes a protein HBX. HBX inhabits the rejoining of DDB1 with DNA. In the DNA repair system, DBB1 is very important. HBX reduces the repairing capability of the DNA repair system. The EST analysis show a lower expression of DNA repair genes in HCC. A low frequency of HCC was observed in mousse with over expression of MGMT, a DNA repair gene. These suggest that HBV may induce HCC by inhabiting the DNA repair system. The DNA repair genes play a import role in the pathogenesis of HCC.DNA repair system continuously monitor chromosome to protect the integrity of DNA. There four main DNA repair pathways. The base excision repair (BER) pathway is one of them. BER excise and replace damaged DNA base. DNA glycosidase initiate this process by releasing the modified base. This is followed by cleavage of the sugar-phosphate chain, excision of the basic residue, and local DNA synthesis and ligation.hOGG1 gene and MB04 gene are among the genes of glycosidase. Study on the association between HCC and these two genes may help us to better understand the pathogenesis of HCC, and provide theoretical ways to prevent, diagnose and treat HCC.Material and Method1. Material96 HCC patients and 96 healthy controls were enlisted. All patient and healthy controls were enlisted. All patients and healthy controls come from Henan. The data such as smoking, alcohol consumption and HBV infection were recorded. 5ml ofperipheral blood was taken from each patient and healthy control. The blood sample were mixed with sodium citrate and preserved at -20℃.2. MethodAfter DNA was extracted from the blood sample,Ser326Cys and Clu346Lys,the polymorphic Loci of hOGG1 and MBD4 gene, were amplified by Polymerase Chain reactions (PCR). after the application was identified by gel electrophoresis, the amplified DNA strips were purified. Then the Ser326Cys and Clu346Lys polymorphisms in the two groups were detected by DNA analysis. Polyphred, a software, was used to analysis and determine the association between these genetic polymorphisms and risk of HCC.Protocol:Peripheral blood→ DNA extraction→ PCR application→Gel electrophoresis→ Purification→ DNA analysis3. Statistic analysisThe relative risk was expresses as odds rations (ORs) and 95% confidence intervals (CIs).All statistic tests were two sided. Statistic Analysis (SAS,SAS institute, Cary, NC, USA) was used.Results3 genotypes (Ser/Ser, Cys/Ser, and Cys/Cys) were found in exon 7 of the hOGGi. The frequency of allelic hOGGi gene in HCC patients were high than that of control group (43% vs 3 Irrespectively). In the HCC group, the frequencies of Cys/Cys, Cys/Ser , and Ser/Ser were 20.9%,44.2%,and 34.9% respectively. Compared with the control group, the frequency of genotype Cys/Cys and Cys/Ser were lower than in the HCC group, while the Ser/Ser genotype was higher, it was show, the risk of HCC were about 2 times higher in patient with Cys/Cys genotype ( OR=1.9). The frequency of allele Cys in the HCC group was significantly higher than in the control group (43% vs 33.1% respectively)3 genotypes (Glu/ Glu, Glu /Lys, and Lys / Lys) were found in exon 3 of the MBD4 gene. In the control group the frenquency of genotype Glu/ Glu, Glu /Lys, and Lys / Lys were 40.4%,46.8%,12.8% respectively while in the HCC group. They were 41.7%,48.8% and 9.5%. There were no significant difference in the frequency of更多还原