【作者】 刘冰；

【导师】 陈坤；

【作者基本信息】 浙江大学 ， 劳动卫生与环境卫生学， 2010， 博士

【摘要】 研究背景及目的结直肠癌的发病率和死亡率在全球仍持续增加。据估计,2007年结直肠癌的新发病例数接近120万,死亡63万,分别比2000年增加了27%和28%。虽然我国属于结直肠癌的传统低发区,但近年来我国结直肠癌的发病率也呈明显上升趋势,根据中国国家癌症数据库资料表明,结直肠癌是中国1991～2005年期间发病率上升速度最快的第三大恶性肿瘤,仅次于肺癌、女性乳腺癌。对结直肠癌的病因学研究表明,结直肠癌的发生是一个多因素、多阶段的过程,是由环境因素和遗传因素综合作用的结果。环境因素对于结直肠癌的形成和进展起到了重要的作用,其至少可以解释约70%的散发性结直肠癌的发生,饮食和生活方式被认为与结直肠癌的发病尤为密切。然而接触同样的环境暴露,并不是每一个个体都会发病,由基因多态决定的个体肿瘤易感性的不同是导致结直肠癌发生的遗传基础。目前发现,许多肿瘤的发生并非单纯由细胞无限增殖所引起,细胞凋亡途径受阻或凋亡功能丧失已成为一些肿瘤发生的重要诱因,而由Caspase家族介导的凋亡是机体最主要的凋亡方式,可能成为影响结直肠癌易感性的潜在遗传因素。因此,本次研究以我国结直肠癌高发区浙江省嘉善县为研究现场,开展以自然人群为基础的病例对照研究,通过传统流行病学和分子流行病学的有机结合,全面分析影响结直肠癌发病风险的环境暴露因素和Caspase凋亡通路基因多态性,深入探讨基因与环境因素之间的潜在交互作用,为结直肠癌的病因学研究提供流行病学线索,并为结直肠癌的人群防治和干预措施的制定提供科学依据。材料和方法研究以浙江省嘉善县作为研究现场,采用以人群为基础的病例-对照研究设计。在2002-2008年间,根据嘉善县肿瘤登记所的监测资料收集结直肠癌存活病例498例,采用随机抽样的方法从健康人群中抽取无肿瘤患病史的嘉善县常住居民838例作为对照。现场调查阶段获取研究对象的调查问卷资料和血样；实验设计阶段以候选功能基因多态策略和Tag SNP策略综合筛选Caspase凋亡通路上的基因多态；实验检测阶段应用改良盐析法提取DNA,以PCR-RFLP法检测拟研究的基因多态；数据分析阶段,联合应用单因素Logistic回归分析、叉生分析、分层分析、多因子降维分析、分类回归树分析等多种方法及模型,评估环境暴露、Caspase凋亡通路上基因多态的主效应、基因—基因、基因—环境联合效应与结直肠癌发病风险的关联。结果病例组和对照组的人口学特征分布均衡,肿瘤家族史在两组间分布无统计学差异。未发现饮酒相关变量与结直肠癌发病存在统计学关联。吸烟但不吸入烟雾者相比吸烟且吸入烟雾人群,结直肠癌的发病风险降低(OR=0.41,95%CI：0.24-0.70),但是非吸烟人群与烟雾吸入的吸烟人群相比结直肠癌的风险效应值无统计学意义(OR=1.22,95%CI：0.86-1.72)。随着平均吸烟量的增加,结直肠癌的发病风险也相应增加。但是未发现累积吸烟量与结直肠癌发病风险存在统计学关联。饮茶对于结直肠癌的发病风险呈保护性效应,其效应值为0.49(95%CI：0.36-0.66)。饮茶量的增加与结直肠癌的发病风险呈剂量反应关系(P trend=0.05),年均饮茶量超过3kg的个体,其结直肠癌的发病风险可降低69%(OR=0.31,95%CI：0.18-0.54)。饮食也与结直肠癌的发生存在显著关联,红肉摄入量大于19.45kg／年的个体,相比参照组小于等于4.2kg／年的个体,结直肠癌的发病风险可降低36%(OR=0.64,95%CI：0.45-0.91)。白肉的摄入量与结直肠癌的发生呈反向关联,年摄入量大于2.4kg以上剂量组,均可显著降低结直肠癌的发生风险。红烧鱼的摄入则可增加结直肠癌的发病风险,年摄入量超过7.2kg的个体发生结直肠癌的风险是参照组的1.64倍(95%CI：1.17-2.30)。对Caspase凋亡通路相关基因多态与结直肠癌关联的主效应分析表明Caspase3 rs4647693 AA基因型对结直肠癌易感性具有保护效应,Caspase8 rs3769818和rs3834129两多态的单体型A-Ins可显著增加直肠癌的发病风险。以环境因素分层,发现在不饮酒人群中,Caspase8 rs3769818 GA携带者相比GG携带者结直肠癌的发病风险增加了32%(OR=1.32,95%CI：1.00-1.74)；Caspase3rs4647693 AA携带者相比GG携带者结直肠癌的发病风险降低了58%(OR=0.42,95%CI：0.19-0.93);Caspase3 rs2696056 GC携带者较GG携带者则增加了42%(OR=1.42,95%CI：1.02-1.96)的发病风险。基因-基因的两因素交互分析结果表明Caspase8 rs3769818和Caspase3 rs4647693的多态基因型存在协同效应,但是交互效应值无统计学意义(OR=0.71,95%CI：0.44-1.15)。Caspase8 rs3769818 A等位基因和Caspase3 rs12108497 C等位基因者相比于同时携带Caspase8 rs3769818GG和Caspase3 rs12108497 C等位基因的个体,患结直肠癌的风险增加了45%(OR=1.45,95%CI：1.05-2.02),但是上述两多态的交互效应也无统计学意义(OR=0.67,95%CI：0.42-1.06)。应用MDR和CART两种软件探索结直肠癌发生的基因-基因、基因-环境高阶交互效应模型,结果表明环境因素对结直肠癌的发生居主导作用,Caspase凋亡通路上的低外显性基因多态对结直肠癌的发生贡献较小,它们可能以凋亡基因Caspase3-DNA修复基因PARP1-细胞周期调控基因p21的协同作用模式参与结直肠癌的发生发展。结论环境因素特别是饮食是嘉善县居民结直肠癌发生的主要影响因素,Caspase3 Tag SNP rs4647693通过隐性效应模式降低了个体对结直肠癌的遗传易感性。在基因-环境的交互作用中,仍以环境暴露对结直肠癌发病风险起主导作用。研究结果尚需大样本、多中心的流行病学研究予以验证。更多还原

【Abstract】 Backgrouds and ObjectivesThe incidence and mortality of colorectal cancer show increasing tendency worldwide. It was estimated that the new cases in 2007 would be approximate 1 200 000 and the death cases would be 630 000, a total increase of 27% and 28% respectively, compared with the figures in 2000. While the incidence tends to be low in China, however, during the past decades, there has been remarkable increase in the incidence of colorectal cancer. According to the Chinese National Cancer Database of 2003, colorectal cancer is one of the three cancers with most rapidly increasing incidence in the country between 1991 and 2003, and finally it ranks the third commonest malignant tumor, just behind lung cancer and female breast cancer.Most common human cancers, including colorectal cancer, have a multifactorial etiology involving complex interplay of genetic susceptibility and environmental exposures. It is widely accepted that environmental factors play key roles in the development and progression of colorectal cancer. The proportion of colorectal cancer attributed to environmental factors has been estimated to be above 70%. Diet and lifestyles are considered to be intimately associated with colorectal cancer risk. However, exposure to the same environmental factors, not everyone will develop colorectal cancer. Genetic polymorphisms have a key role in individual predisposition to colorectal cancer. It was found that progressive inhibition or evasion of apoptosis has been found during the transformation of colorectal epithelium to carcinoma, indicating that dysfunction of apoptosis has an important role in colorectal tumorigenesis. Apoptosis, which is mainly mediated by caspase family, play an important role in the maintenance of tissue homeostasis and inhibition of tumor formation in organisms. Apoptotic capacity is a subject of significant interindividual variations, which are largely attributed to hereditary traits. Genetic polymorphisms located within cell death genes may influence apoptosis activity. Low activity of apoptosis would favor cancer development because of the failure to eliminate cellular clones carrying DNA damage and propensity to inflammation.In order to explore the effect of environmental exposure, genetic polymorphisms in caspase mediated apoptosis pathway as well as the interactions between selected genes and environmental factors on colorectal cancer risk, a population-based case-control study was conducted in Jiashan County, Zhejiang Province, of which both field questionnaire survey and laboratory genotyping detection were adopted.Materials and MethodsFrom May 1989 to April 1990, a prospective cohort study based on a colorectal cancer survey was initiated in Jiashan County, Zhejiang Province, China. Subsequently, a cancer surveillance and registry system covering the whole population in Jiashan County was established to report new patients with various cancers. Between 2002 and 2008,498 CRC cases had been recruited in this study, based on the surveillance and registry system. All of the CRC cases were confirmed by histological diagnosis. However, patients with other malignant disease in their medical history were excluded from this study. Simultaneously,838 controls without the history of cancer were selected randomly from the corresponding population. All participants were Han Chinese residents.During the field survey, a structured questionnaire, including demographic characteristics, personal habits (cigarette smoking, alcohol drinking, etc) and health factors (family history of cancer at any site including all first- and second-degree relatives of both genders, medical and dietary history, etc) and 5 ml venous blood sample were collected from each subject. A combinative strategy of functional genetic polymorphisms and tagging singlenucleotide polymorphisms was applied to the selection of susceptible biomarker in apoptosis mediated pathway. Tagging single-nucleotide polymorphisms were selected by searching Han Chinese data from the HapMap project using the Tagger program. The genomic DNA was extracted from peripheral blood samples using modified salting-out procedure. For determination of the selected genetic polymorphisms, Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) assay was performed. Multiple statistical methods were used when estimating the major effect and the interactive effect of environmental factors and the genetic polymorphisms involved in caspase mediated apoptosis pathway, including Logistic regression, stratified analysis, crossover analysis, Multifactor Dimensionality Reduction (MDR) analysis and Classification And Regression Tree (CART) analysis.ResultsThe demographic characteristics and family history of cancer did not differ significantly between cases and controls. There was no association between variables concerning drinking and colorectal cancer risk. Smokers without smoke inhalation had lower risk of suffering from colorectal cancer compared with smoke inhalation smokers (OR=0.41, 95%CI:0.24-0.70), while nonsmokers did not show significant difference in colorectal cancer risk when compared with smoke inhalation smokers (OR= 1.22,95%CI:0.86-1.72). An increased risk of colorectal cancer was indicated by the higher daily cigarette consumption of smoking. But the association between the accumulated cigarette consumption and colorectal cancer risk was not significant. Tea drinking could significantly decrease the colorectal cancer risk about 51%(OR=0.49,95%CI: 0.36-0.66). Significant dose-response relationships were found for the average amount of tea consumed by year (P trend=0.01). The reduction in risk was most evident among those who consume tea above 3kg a year (OR=0.31,95%CI:0.18-0.54). Diet showed strong association with colorectal cancer risk. Individuals who consumed red meat more than 19.45kg every year, has a significant 36% decreased risk of colorectal cancer (OR=0.64,95%CI:0.45-0.91), compared with individuals in the lowest category. Higher chicken consumption showed a protective effect on colorectal cancer risk. While source-boiled fish has a significantly positive association with colorectal cancer. The risk of suffering colorectal cancer of people who consumed source-boiled fish more than 7.2kg one year is 1.64 times higher than the reference group (95%CI:1.17-2.30). In this study, totally 12 genetic polymorphisms of 5 genes involved in caspase mediated apoptosis pathway were analyzed, of which eight were potential functional genetic polymorphisms and four are tagging single nucleotide polymorphisms. It was found that Caspase3 rs4647693 AA genotype had a protective effect on susceptibility to colorectal cancer (OR=0.50,95%CI:0.26-0.95). The carriers with haplotype A-Ins in Caspase8 rs3769818 and rs3834129 showed an increased risk of rectal cancer (OR=1.28,95%CI: 1.00-1.63). Among the non-drinkers, it was found that compared with Caspase8 rs3769818 GG genotype carriers, the GA genotype carriers had a 32% increased risk of colorectal cancer (OR=1.32,95%CI:1.00-1.74); the AA genotype of rs4647693 in the Caspase3 gene was associated with decreased risk compared with homozygotes of the major alleles (OR=0.42,95%CI:0.19-0.93); the rs2696056 GC genotype in Caspase3 gene was significantly associated with increased cancer risk compared with the GG genotype (OR=1.42,95%CI:1.02-1.96). Crossover analysis suggested that Caspase8 rs3769818 had combined effects with Caspase3 rs4647693 GG risk genotype. Combinative effect was also observed between Caspase8 rs3769818 and Caspase3 rs12108497. However, neither of the interactions between the SNPs had statistical significance.High order interaction models were explored by MDR and CART methods. Both of the two data-mining methods indicated that environmental factors played an important role in the colorectal cancer risk. The selected genetic polymorphisms may further modify the colorectal cancer risk but not as a main effect.ConclusionsOur findings verified the important influence of environmental factors on CRC risk. Caspase3 rs4647693 has an independent effect on colorectal cancer risk with recessive model. Further studies with large sample size are warranted to validate our findings.更多还原