【作者】 黄金梅；

【导师】 曾小云；

【作者基本信息】 广西医科大学 ， 流行病与卫生统计学， 2011， 硕士

【摘要】 肝细胞癌(hepatocellular carcinoma, HCC,以下简称肝癌),是世界上最常见的恶性肿瘤之一,高居世界肿瘤死因谱第三位,严重威胁着人们的健康与生命。我国是肝癌高发国家之一,广西是我国肝癌高发区,其死亡率约为34.74/10万,位于广西肿瘤死因谱首位。肝癌病程短,病死率高,目前无有效延长病人生存时间的治疗方法,因此,肝癌的一、二级预防成了急需解决的重要公共卫生问题。肝癌病因复杂,是遗传和环境因素相互作用的结果,而且其发生、发展具有明显的种族、地域及个体差异。以往研究结果显示,肝癌的主要致病因素有:病毒性肝炎感染、食物中黄曲霉毒素污染、饮用水藻类毒素污染以及吸烟饮酒等生活不良习惯等。但是,即使生活在肝癌致病因素高暴露的人群中,也只有少部分人最终发展为肝癌,不同遗传多态性的个体对环境致病因素的易感性不同,且易感基因本身不足以引起疾病,或者说在疾病发生过程中只起到“微效”作用,遗传因素与特定的环境因素相结合,才能更好的解释肝癌发生的机制。环境中的致癌物进入人体后在多种酶的作用下,经过生物转化最终形成终致癌物,从而对其主要靶器官产生慢性毒作用,而DNA修复酶则在防止基因突变方面具有重要作用。研究显示,许多DNA修复酶基因多态性可能与肝癌易感性存在一定的相关性,如X射线损伤交叉互补修复酶1基因(XRCC1)、着色性干皮病基因D(XPD)、人8-羟基鸟嘌吟糖苷酶1(hOGG1)等。近年来,关于DNA修复酶基因多态性、基因-环境对肝癌发生的影响已广泛引起重视,但至今尚无定论,研究结果也不一致。本研究选择参与DNA损伤修复通路的碱基切除修复(base excision repair, BER)的XRCC1基因,应用单纯病例研究方法,探讨XRCC1基因与某些环境危险因素交互作用对肝癌发生风险的影响,同时运Meta分析方法,对XRCC1基因多态性与肝癌发病风险相关的文献研究结果进行合成分析和定量合并,探讨XRCC1基因多态性与肝癌发生的相关性。第一部分XRCC1基因多态性与环境因素在肝细胞癌发生中交互作用的单纯病例研究目的:探讨某些环境暴露因素与XRCC1基因多态性的交互作用对肝癌发生危险性的影响。方法:采用以医院为基础的单纯病例研究方法。病例来源于2007年6月～2010年12月在广西医科大学第一附属医院和广西中医学院第一附属医院住院的所有同意参与调查并经临床相关诊断,未接受过化疗和放射治疗的新发肝癌患者共500例,采用课题组设计的流行病学调查问卷,经由专门培训的调查员对研究对象进行调查,内容包括研究对象基本情况、既往史、个人史、家族史、吸烟史和饮酒史等资料,并收集病人体格检查和实验室检查结果。采集研究对象4ml外周血,以酶联免疫吸附法(Enzyme-linked immunosorbent assay, ELISA)对其进行HBV感染五项指标和HCV抗体检测;以酚-氯仿法提取全基因组的DNA,采用TaqMan MGB实时荧光定量聚合酶链式反应(Real-time quantitative polymerase chain reaction, RT-PCR)技术检测XRCC1基因单核苷酸多态性。应用SPSS13.0 for windows统计软件对所收集的数据进行统计分析,运用二分类Logistic回归模型进行基因-环境、基因-基因的交互作用效应分析,用比值比(odds ratio, OR)及其95%可信区间(confidence interval, CI)表示关联强度。Hardy-Weinberg遗传平衡吻合度检验应用SHEsis软件。结果:(1)研究对象一般情况:研究对象按照XRCC1基因的野生型和突变型分类,两组之间年龄、性别、民族、职业和婚姻状况等方面差异均无统计学意义(P>0.05)。(2)基因-环境交互作用:XRCC1-280与e抗体对肝癌的发生有正相乘交互作用(校正后,P=0.047,OR=1.809,95%CI为1.007～3.249);XRCC1-194与饮酒对肝癌的发生有正相乘交互作用(校正后,P=0.041,OR=1.496,95%CI为1.016～2.204);XRCC1-194与表面抗体阳性对肝癌的发生有负相乘交互作用(校正后,P=0.045,OR=0.0576,95%CI为0.336～0.987)。XRCC1三个位点的突变等位基因与吸烟、慢性HBV感染、食鱼生史、家族史,肝炎病史、核心抗体、e抗原、抗HCV、AFP等之间均未见交互作用。(3)基因-基因交互作用:XRCC1突变等位基因对肝癌具有相乘交互作用。同时携带至少一个XRCC1-194(CT/TT)突变等位基因和至少一个XRCC1-28(0GA/AA)突变等位基因的个体肝癌发病风险增加2.595倍(ORadj = 3.595)。同时携带至少一个XRCC1-194(CT/TT)突变等位基因和至少一个XRCC1-399(GA/AA)突变等位基因的个体肝癌发病风险增加1.790倍(ORadj = 2.790)。同时携带至少一个XRCC1-280(GA/AA)突变等位基因和至少一个XRCC1-399(GA/AA)突变等位基因的个体肝癌发病风险增加1.310倍(ORadj = 2.310)。结论:(1)携带XRCC1-194突变等位基因与饮酒对肝癌可能存在交互作用;携带XRCC1-194突变等位基因与表面抗体对肝癌可能存在交互作用。(2)携带XRCC1-280突变等位基因与e抗体对肝癌可能存在交互作用。(3)同时携带XRCC1-194,XRCC1-280和XRCC1-399任意两个的突变等位基因对肝癌的发生可能有交互作用。第二部分XRCC1基因多态性与肝细胞癌易感性关系的Meta分析目的:探讨XRCC1基因多态性与肝细胞癌易感性的关系。方法:以“hepatocellular carcinoma”或“HCC”,“XRCC1”或“X-ray repair cross-comple-menting group 1”,“genetic polymorphism”或“SNP”等为检索词检索PubMed、Medline、Embase等英文数据库;以“肝癌”、“肝细胞癌”或“肝肿瘤”,“多态性”,“X线修复交叉互补基因1”等为检索词检索清华同方、维普中文科技期刊、万方数据库、中国生物医学文献等中文数据库,获得有关XRCC1基因多态性与肝细胞癌发病风险的病例对照研究资料,Meta分析采用Review Manager 5.0软件和Stata10.0软件,对纳入的各研究数据进行统计处理及异质性检验,计算合并的OR值及OR 95%CI。结果:通过文献检索和筛选,最终纳入的文献中:对于XRCC1-399,共包括8篇文章的研究,涉及1604例肝癌患者和2185例对照个体;对于XRCC1-280,有3篇文章进行了研究,涉及700例肝癌患者和678例对照个体;对XRCC1-194,共包括3篇文章的研究,涉及663例肝癌患者和761例对照个体。将三种基因型数据分别用四种遗传模型分组方式进行Meta分析。显性模型下,XRCC1-399合并的OR=0.99,95%CI=0.75～1.31,XRCC1-280合并的OR=0.98,95%CI=0.74～1.28 , XRCC1-194合并的OR=0.98,95%CI=0.79～1.22。结论:XRCC1-199、XRCC1-280和XRCC1-399基因多态性与肝细胞癌易感性的关联没有统计学意义。更多还原

【Abstract】 Hepatocellular Carcinoma (hereafter referred to as HCC) is one of the most common malignant tumors in the world. Ranking 3rd place on the world’s rumor list by morality rate, it has great threat on peoples’health and life.With an HCC incidence rate of 30.3 out of every 100,000 people, China makes one of the countries with high HCC occurrence. Its mortality rate in Guangxi, an HCC high-risk area, is 27.31 out of every 100,000 people, ranking HCC top on the province’s rumor list by mortality rate. HCC has short clinical course as well as high morality rate, and there is not yet any effective therapy to extend HCC patients’lives. Therefore, primary and secondary HCC prevention has become an urgent and important public health issue.Pathogeny of HCC is very complicated, as it results from the interactions of genetic and environmental factors and varies by race, region and individual. Previous studies have shown that HCC was mainly caused by infection of virus hepatitis, aflatoxin contamination in food, algal toxin contamination in drinking water, improper living habits such as smoking and drinking, etc. However, even in the HCC high-risk group, only a small part get infected. Genetic polymorphism leads to different susceptibilities of environmental pathogenic factors, and susceptibility genes only have limited pathogenic effect during disease occurrence. Only by combining genetic and specific environmental factors can we explain the mechanism of HCC. After entering human body from the environment, carcinogen develops into ultimate carcinogen by biotransformation under the function of various enzymes, and hence brings out chronic toxic effects on its main target organ; while DNA repair enzyme plays an important role in prevention of genetic mutation. Studies reveal certain relationship between gene polymorphism of many DNA repair enzymes and susceptibility of HCC, Such as X-ray repair cross-complementing (XRCC1), Xeroderma pigmentosum group D(XPD), human 8-oxoguanine glycosylase 1(hOGG1),etc. In the recent years, gene polymorphism of DNA repair enzymes and impacts of gene-environment interactions on HCC occurrence have attracted broad attention, but there hasn’t been any final conclusion due to inconsistent research results.This study is based on XRCC1 gene, which participates in base excision repair (BER) of DNA repair pathways, to explore on impacts of interactions between XRCC1 and certain environmental risk factors on HCC occurrence risk with case-only study method, and to discuss the correlation between XRCC1 gene polymorphism and HCC occurrence by conducting synthesis analysis and quantitative combination on documentary study results relevant to XRCC1 gene polymorphism and HCC occurrence with meta analysis method. Part 1 Case-only Study on Interactions between XRCC1 Gene and Specific Environmental Factors during HCC OccurrenceObjective To explore the impacts of interactions between environmental exposure factors and genetic polymorphism in XRCC1 on risk of HCC.Methods The research was designed by a case-only study based on hospitals. Studied cases are the 500 new HCC cases in The First Affiliated Hospital of Guangxi Medical University and The First Affiliated Hospital of Guangxi Traditional Chinese Medical University during June 2007 to December 2010, who agreed to attend the survey, received relevant clinical diagnosis but never accepted chemotherapy and radiation.This study adopts epidemiological questionnaire. Professional surveyors surveyed on various aspects of the research objects, including their basic condition, previous history, personal history, family history, smoking history, drinking history, etc. and collected their physical examination and laboratory examination results. Surveyors collected 4ml peripheral blood of each study subject to conduct the examinations on 5 indexes for HBV diagnosis and Anti-HCV by enzyme-linked immunosorbent assay (ELISA); extracted entire genomic DNA with phenol-chloroform extracting method to detect single nucleotide polymorphism of XRCC1 gene with TaqMan MGB Real-time quantitative polymerase chain reaction (RT-PCR); conducted statistical analysis on the data with the statistical software called“SPSS13.0 for Windows”, using binary logistic regression model to analyze gene-environment and gene-gene interactions and indicating relative risk with odds ratio (OR) and its 95% confidence interval (CI).Results (1)General conditions of study subjects: The two groups are divided by wild-type and mutant-type of XRCC1 gene, so differences in their age, gender, nationality and vocation have no statistical significance(P>0.05). (2)Gene-environment interactions: XRCC1-280 and Anti-Hbe have positive multiplicative interaction on HCC occurrence(after adjusting, P=0.047, OR=1.809,95%CI=1.007~3.249);XRCC1-194 and drinking alcohol, multipli- cative interaction on HCC occurrence(after adjusting ,P=0.041,OR=1.496, 95%CI=1.016~2.204); XRCC1-194 and HBsAb have negative multiplicative interaction on HCC occurrence(after adjusting, P=0.045,OR=0.0576, 95%CI= 0.336~0.987). Multiplicative interactions are not found between mutant alleles and smoking, chronic HBV infection, raw-fish eating history, family history,HBeAg, HBcAb, Anti-HCV, AFP, etc. (3) gene-gene interaction: XRCC1 mutant allele had interaction on HCC. When carrying at least one XRCC1-194 (CT / TT) mutant allele and at least one XRCC1-280 (GA / AA) Mutant allele ,it would have 2.595-fold increased risk of HCC (ORadj = 3.595). When carrying at least one XRCC1-194 (CT / TT) mutant allele and at least one XRCC1-399 (GA / AA) mutant allele, it would have 1.790-fold increased risk of HCC (ORadj = 2.790). When carrying at least one XRCC1-280 (GA / AA) mutant allele and at least one XRCC1-399 (GA / AA) mutant allele ,it would have 1.310-fold increased risk of HCC (ORadj = 2.310).Conclusions (1) Carrying XRCC1-194 mutant allele and drinking may have an interaction related with HCC; carrying XRCC1-194 the mutant allele and HbsAb would have an interaction related with HCC.(2) Carrying XRCC1-280 mutant allele and HbeAb may have an interaction related with HCC(3) While carrying XRCC1-194, XRCC1-280 and XRCC1-399 mutant alleles of any two may have multiplied interaction related HCC. Part 2 Meta-Analysis on Relationship between XRCC1 Gene Polymorphisms and Susceptibility to Hepatocellular CarcinomaObjective To explore the XRCC1 gene polymorphism and susceptibility to hepatocellular carcinoma.Methods To obtain case-control study information about XRCC1 gene poly- morphisms and hepatocellular carcinoma occurrence risk by searching English databases such as PubMed, Medline and Embase with terms including“hepatocellular carcinoma”(or“HCC”),“XRCC1”(or“X-ray repair cross- comple-menting group 1”“genetic polymorphism”(or“SNP”), etc. , and searching Chinese databases such as CNKI, VIP, Wanfang Data and CBM with terms including“ganai”,“ganxibaoai”,“ganzhongliu”,“duotaixing”,“X xian xiufujiaochahubujiyin 1”in chinese;As for Meta-Analysis, to calculate combined OR value and its 95% CI by conducting combinatorial statistics and heteroge- neity test on the data collected with software Review Manager5.0 and Stata10.0.Results After literature searching and screening, the following information is ultimately selected: for the XRCC1-399, a total of eight articles of independent study, involving 1604 HCC cases and 2185 controls; for the XRCC1-280, three articles, involving 700 cases and 678 controls; and for the XRCC1-194, three articles, involving 663 cases and 761 controls. Meta-Analysis was conducted on the three genotype data by grouping in four genetic models. The pooled OR for XRCC1-399、XRCC1-280 and XRCC1-194 were 0.99(95%CI:0.75~1.31)、0.98 (95%CI:0.74~ 1.28) and 0.98 (95%CI:0.79~1.22) in the dominance model.Conclusions XRCC1-199, XRCC1-280 and XRCC1-399 polymorphism may be unrelated to the risk of hepatocellular carcinoma occurrence.更多还原