【作者】 于宏杰；

【导师】 徐飚； 付朝伟；

【作者基本信息】 复旦大学 ， 流行病与卫生统计学， 2009， 硕士

【摘要】 【目的】分析环境因素、遗传因素及其交互作用在食管鳞癌发生中的作用,探讨食管癌相关基因MGMT（O⁶-methylguanine-DNA methvltransferase,O⁶-甲基鸟嘌呤-DNA甲基转移酶）和XRCCl（X-ray repair cross-complementing gene l,X射线交错互补修复基因1）的蛋白表达情况其与食管癌发生、相关临床特征和基因多态间的关系。【方法】以食管癌高发区江苏省扬中市作为研究现场,采用以人群为基础的病例对照研究设计,分析吸烟、饮酒、饮水、食用陈米等环境凶素与食管鳞癌发生之间的关系。应用PCR-RFLP等分子生物学技术分析DNA损伤修复基因XRCCl的三个常见的基因多态Arg194Trp、Arg280His和Arg399GIn及DNA甲基转移酶基因MGMT的Ile143Val和Lys178Arg多态在食管鳞癌发生中的作用及其与环境因素之间的交互作用。应用免疫组化（IHC）方法检测XRCCl和MGMT在食管癌患者和正常人群中的表达情况,探讨相蛋白表达与食管癌临床特征和基因多态间可能存在的联系。食管癌病例均来自扬中市肿瘤登记报告系统,为2004年1月1日至2008年5日14日间新发并经病理学确珍断的食管鳞状细胞癌患者,年龄大于30岁且在扬中市生活5年或以上。愿意参加调查并签署知情同意书者,在确诊后2～3周内进行流行病学问卷调查,并收集空复静脉血标本5ml。对照来自与病例同期的扬中市居民,通过扬中市常住居民名单随机选取,要求对照年龄在30岁以上,身体健康,无肿瘤病史及胃十二指肠溃疡史,均为愿意参加调查并签署知情同意书者。剪取手术患者的癌中心组织,并从胃镜室集正常健康成人的食管粘膜标本。数据录入与分析分别采用EpiData3.0和SPSS 11.0（Chicago,Illinois,USA）、STATA 9.2（Texas,USA）统计软件,应用单因素x²检验和多因素非条件Logistic回归模型分析环境因素、基因型和表型与食管鳞癌之间的关系。【结果】自2004年1月1日至2008年5月14日,共收集食管鳞癌病例432例,平均年龄61.5±7.8岁,男性61.8%,女性38.2%:对照915例,平均年龄57.05±9.05岁,男性49.3%,女性50.7%。在按性别进行分层并调整了年龄（连续）和受教育程度后发现,与饮用自来水者相比,饮用河沟水能显著增加男女性患食管癌的危险,其中男性OR=4.20（95%CI:2.90-6.07）,女性OR=8.37（95%CI:5.09-13.75）。陈米摄入能增加女性患食管癌的危险（OR=12.78,95%CI:2.69-60.69）,但在男性中未发现食管癌与食用陈米间的关系。未观察到吸烟与食管癌发生间的关系,但吸烟者有食管癌发病危险增加的倾向。尽管未发现女性饮酒与食管癌间的关系,但男性饮酒者患食管癌的危险显著增加（OR=2.03,95%CI:1.43-2.89）。XRCC1 Arg194Trp位点194T等位基因在食管癌病例组和对照组中的频率分别是33.0%31.3%。与野生型CC相比,杂合型CT和纯合突变型TT的比值比OR分别为1.18（95%CI:0.80-1.73）和1.06（95%CI:0.84-1.36）,将突变型CT和TT合并后,OR为1.09（95%CI:0.86-1.37）;经性别、年龄（连续）、受教育程度、吸烟史、饮洒史、陈米摄入史和河沟水饮用史调整后,杂合型CT和纯合突变型TT的比值比OR分别为1.19（95%CI:0.76-1.84）和1.06（95%CI:0.81-1.39）,将和TT合并后,OR为1.08（95%CI:0.84-1.40）。XRCC1 Arg280His位点280A等位基因在食管癌病例组和照组中的频率分别是11.0%和14.4%。与野生型GG相比,杂合型GA和纯合突变型AA的OR值分别为0.76（95%CI:0.31-1.83）和0.70（95%CI:0.52-0.92）,将突变型GA和AA合并后,OR为0.70（95%CI:0.53-0.92）.纯合突变型AA和合并后的突变型GA+AA均与较低食管癌的罹患风险有关:经性别、年龄（连续）、受教育程度、吸烟史、饮酒史、陈米摄入史和河沟沟水饮用史调整后,杂合型GA和纯合突变型AA的比值比OR分别为0.94（95%CI:0.36-2.48）和0.80（95%CI:0.58-1.10）,将GA和AA合并后,OR为0.81（95%CI:0.60-1.11）。XRCC1 Arg399Gln位点399A等位基因在食管癌病例组和对照组中的频率分别是27.1%和23.3%。与野生型GG相比,杂合型GA和纯合突变型AA的OR分别为1.84（95%CI:1.17-2.88）和1.07（95%CI:0.84-1.36）,将GA和AA合并后,OR为1.16（95%CI:0.92-1.47）;经性别、圻龄（连续）、受教育程度、吸烟史、饮酒史、陈米摄入史和河沟水饮用史调整后,杂合型GA和纯合突变型AA的OR分别为1.48（95%CI:0.89-2.48）和1.16（95%CI:0.8-1.53）,将GA和AA合并后,OR为1.21(95%CI:0.93-1.561。XRCC1Arg194Trp基因多态与饮河沟水、陈米摄入、吸烟和饮酒的交互效应比值比OR_int分别为0.90（95%CI:0.50-1.62,P=0.720）、0.76（95%CI:0.20-2.86,P=0.684）、0.96（95%CI:0.57-1.62,P=0.874）和0.68（95%CI:0.39-1.16,P=0.157）,未见XRCC1 Arg194Trp基因多态与环境暴露因素间存在有统计学意义的相乘交互效应。XRCC1 Arg280His基因多态与饮河沟水、陈米摄入、吸烟和饮酒的交互效应比值比OR_int分别为0.84（95%CI:0.42-1.69,P=0.628）、1.11（95%CI:0.22-5.63,P=0.897）、1.23（95%CI:0.65-2.32,P=0.530）和0.96（95%CI:0.50-1.85,P=0.895）,未见XRCC1 Arg280His基因多态与环境暴露因素间存在有统计学意义的相乘交互效应。XRCC1 Arg399Gln基因多态与饮河沟水、陈米摄入、吸烟和饮洒的交互效应比值比OR_int分别为1.50（95%CI:0.82-2.74,,P=0.187）、6.58（95%CI:1.60-27.11,P=0.009）、1.02（95%CI:0.60-1.73,P=0.955）和1.46（95%CI:0.85-2.53,P=0.175）,XRCC1 Arg399Gln)基因多态与陈米摄入间存在相乘交互效应。经性别、年龄（连续）、受教育程度、吸烟史、饮酒史、陈米摄入史和河沟水饮用史调整后,与XRCC1基因Arg194Trp CC野生型和Arg280His GG野生型相比,同时携带Arg194Trp CC野生型和Arg280His突变型（GA或AA）者患食管癌的风险增加（OR=1.87,95%CI:1.10-3.18,P=0.020）。未见XRCC1 Arg194Trp和Arg399Gln及Arg280His和Arg399Gln基因多态间存在有统计学意义的联系。MGMT基因Ile143Val和Lys178Arg两位点多态性分布完全相同。食管癌病例组和对照组中MGMT基因的G等位基因频率分别为0.6%和0.4%。与野生型AA相比,突变型AG的OR为1.33（95%CI:0.43-4.08）,未发现纯合突变型GG的存在;经性别、年龄（连续）、受教育程度、吸烟史、饮酒史、陈米摄入史和河沟水饮用史调整后,突变型AG的OR为0.79（95%CI:0.23-2.69）。MGMT基因多态与饮河沟水、吸烟和饮酒的交互效应比值比OR_int分别为0.03（95%CI:0.01-0.51,P=0.014）、0.06（95%CI:0.01-0.85,P=0.038）和0.18（95%CI:0.01-2.80,P=0.220）,MGMT基因多态与吸烟和饮河沟水之间存在相乘交互效应。未见MGMT基因多态与饮酒和陈米摄入间存在有统计学意义的相乘交互效应。经性别、年龄（连续）、受教育程度、吸烟史、饮酒史、陈米摄入史和河沟水饮用史永调整后,未见XRCC1和MGMT基因多态间存在有统计学意义的联系。XRCC1在正常对照中的表达阳性率为33.3%（27/81）,在食管癌组织中的表达阳性率为44.3%（62/140）,两者间无显著性差异（x²=2.559,P=0.110）:MGMT在正常对照中的表达阳性率为51.7%（45/87）,在食管癌组织中的表达阳性率为75.2%（98/140）,两者间差异具有显著性（x²=7.688,P=0.006）。MGMT蛋白表达与陈米摄入（P=0.072）和食管癌M分期（P=0.081）间有弱相关性,陈米摄入可能增加MGMT蛋白的表达,而食管癌远处转移可能与MGMT蛋白表达缺失有关。未见XRCC1蛋白表达与性别、年龄、吸烟、饮酒、陈米摄入和饮河沟水及临床病理特征间的关系。未见XRCC1和MGMT基因多态与蛋白表达间存在具有统计学意义的联系。【结论】江苏省扬中市居民食管癌发病危险可能与当地的生活习惯生活习惯有关,饮用河沟水是食管癌发生的重要危险因素;饮酒和食用陈米也能增加食管癌的患病危险,但存在性别差异,饮酒能增加男性管家癌的发病危险,而食用陈米能增加女性食管癌的发病危险。吸烟也有使食管癌发病危险增加的倾向。积极开展健康教痛和健康促进,倡导居民饮用自来水,改变食用陈米的习惯,鼓励戒烟、控制饮酒是预防和控制食管癌的重要措施。MGMT Ile143Val和Lvs178Arg两位点多态性分布相同,未发现XRCC1和MGMT基因多态食管癌发病风险间的关系,但陈米摄入与XRCC1 Arg399Gln多态之间存在相乘交互效应,携带突变基因型（GA或AA）者若食用陈米,则患食管癌的危险增加。吸烟和饮河沟水与MGMT多态之间存在相乘交互效应,携带AA基困型者若吸烟或饮河沟水,则患食管癌的危险增加。另外,XRCC1 Arg280His基因多态与Arg194Trp基因多态间存在交互效应。食管癌的发生可能与MGMT蛋白阳性表达有关,陈米摄入和食管癌远处转移与MGMT蛋白表达间亦有弱相关性,陈米摄入可能增加MGMT蛋白的表达,而食管癌远处转移可能与MGMT蛋白表达缺失有关。更多还原

【Abstract】 [Objective]This study aimed to investigate the role of environmental factors, genetic polymorphisms and protein expressions of XRCC1 and MGMT genes,and their interactions in the risk of esophageal squamous cell carcinoma（ESCC） in a Chinese population.[Methods]A population-based case-control study was conducted in Yangzhong County,Jiangsu Province,China.ESCC cases were diagnosed between 1^st January 2004 and 14^th May 2008 and histologically confirmed,while controls were randomly selected from the local healthy and cancer-free population.Face-to-face interviews were carried out by trained interviewers using a standard structured questionnaire and blood samples were collected after informed consent.Polymerase Chain Reaction （PCR） and Restriction Fragment Length Polymorphism（RFLP） methods were used to detect polymorphisms of XRCC1 and MGMT gene.Histologically confirmed ESCC cases from January 2005 to August 2007 were also collected with paraffin-embedded slides and cancer-free controls were selected with paraffin-embedded slides. Immunohistochemistry assay was conducted with these samples.Epidata 3.0.SPSS 11.0 and STATA 9.2 statistical softwares were used to input and analyze data,and x² test and unconditional logistic regression were occupied to measure the relationship between environmental factors,genetic polymorphisms,protein expression and the possible gene-environment and gene-gene interactions in the risk of esophageal cancer by calculating odds ratio（OR）,adjusted OR（aOR） and 95%confidence interval（95%CI）.[Results]Totally,432 cases（men 61.8%,women 37.2%） were enrolled in this study with the average age of 61.5±7.8 years old and then 915 controls were recruited（men 49.3%,women 50.7%,age in mean of 57.1±9.1 years old）.After stratified by sex and adjusted by age and education years,the results from unconditional logistic regression suggested that river water drinking and alcohol drinking could increase the risk of esophageal cancer with aOR of 4.20（95%CI:2.90-6.07） and 2.03（95%CI:1.43-2.89） in men,respectively,as well as river water drinking and old stocked rice intake with aOR of 8.37（95%CI:5.09-13.75） and 12.78（95%CI:2.69-60.69） in women, respectively. The allele frequencies of XRCC1 Arg194Trp 194T were 33.0%and 31.3%in cases and controls,respectively.Compared with CC genotype,subjects with CT,TT and CT/TTgenotypes got ORs of 1.18（95%CI:0.80-1.73）,1.06（95%CI:0.84-1.36） and 1.09（95%CI:0.86-1.37）,respectively.After adjusted by sex,age,education years, river water drinking,old stocked rice intake,tobacco smoking and alcohol drinking, subjects with CT,TT and CT/TT genotypes had aORs of 1.19（95%CI:0.76-1.84）. 1.06（95%CI:0.81-1.39） and 1.08（95%CI:0.84-1.40）,respectively.The allele frequencies of XRCC1 Arg280His 280A were 11.0%and 14.4%in cases and controls, respectively.Compared with GG genotype,subjects with GA,AA and GA/AA genotypes got ORs of 0.76（95%CI:0.31-1.83）,0.70（95%CI:0.52-0.92） and 0.70 （95%CI:0.53-0.92）,respectively..After adjusted by those factors,individuals with GA,AA and GA/AA genotypes had aORs of 0.94（95%CI:0.36-2.48）,0.80（95%CI: 0.58-1.10） and 0.81（95%CI:0.60-1.11）,respectively.The allele frequencies of XRCC1 Arg399Gln 399A were 27.1%and 23.3%in cases and controls,respectively. Compared with GG genotype,subjects with GA,AA and GA/AA genotypes had ORs of 1.84（95%CI:1.17-2.88） and 1.07（95%CI:0.84-1.36） and 1.16（95%CI:0.92-1.47）. respectively..After adjusted by those factors,individuals with GA,AA and GA/AA genotypes had aORs of 1.48（95%CI:0.89-2.48）,1.16（95%CI:0.88-1.53） and 1.21 （95%CI:0.93-1.56）,respectively.Old stocked rice intake had a positive multiple interaction with XRCC1 Arg399Gln polymorphisms（for interaction,OR=6.58, 95%CI:1.60-27.11,P=0.009）.and subjectls with variant genotypes（GA or AA） had a significant higher risk of ESCC if they took old stocked rice.Compared with wild genotypes of Arg194Trp and Arg280His.subjects with Arg194Trp CC genotype and Arg280His variant genotype（GA or AA） had a significant higher risk of ESCC（for interaction.OR=1.87,95%CI:1.10-3.18,P=0.020）.There was a complete consistency between MGMT Ile143Val and Lys178Arg loci. The allele frequencies of MGMT 143G（178G） were 0.6%and 0.4%in cases and controls,respectively.Compared with AA genotype,subjects with AG genotype got OR of 1.33（95%CI:0.43-4.08）.After adjusted by related factors,subjects with GA genotype got OR of 0.79（95%CI:0.23-2.69）.Tobacco smoking and river water drinking had negative multiple interactions with MGMT polymorphisms,and their ORs were 0.06（95%CI:0.01-0.85,P=0.038） and 0.03（95%CI:0.01-0.51,P=0.014）. respectively,subjects with AA genotype who smoked or drank river water had a significant higher risk of ESCC. Immunohistochemistry analysis detected the XRCC1 positive rates were 44.3% （62/140） and 33.3%（27/81） in cases and controls,respectively,and no significant difference was found（x²=2.559,P =0.110）.The MGMT positive rates were 70.0% （98/140） and 51.7%（45/87） in cases and controls,respectively,and a significant difference was observed（x²=7.688,P=0.006）.There was a weak association between low MGMT protein expression and distant metastasis（P=0.081）,and as also,a weak association between high MGMT protein expression and old stocked rice intake was found（P=0.072）.There were no associations between XRCC1 protein expression and sex,age,tobacco smoking,alcohol drinking,old stocked rice intake,river water drinking and clinical pathological characteristics.Also,there was no association between XRCC1 polymorphisms and XRCC1 protein expression,neither did MGMT polymorphisms and MGMT protein expression.[Conclusions]Common environmental factors like river water drinking,old stocked rice intake,alcohol drinking and genetic factors might contribute to the occurance of ESCC.Tobacco smoking might also play a role in increasing the incidence of ESCC. Drinking tap water,taking fresh rice,smoking cessation and controlling alcohol drinking are important to the prevention of ESCC.There could be modification effects between old stocked rice intake and XRCC1 Arg399Gln,tobacco smoking and MGMT.river water drinking and MGMT,as well as XRCC1 Arg280His and Arg194Trp.High MGMT protein expression might be associated with the risk of ESCC.There might also be a weak association between low MGMT protein expression and distant metastasis as well as a weak association between high MGMT protein expression and old stocked rice intake.更多还原