【作者】 刘友强；

【导师】 王贵英； 于跃明；

【作者基本信息】 河北医科大学 ， 外科学， 2012， 硕士

【摘要】 目的：Survivin是凋亡抑制蛋白(IAP)家族的成员之一，不仅抑制凋亡，也参与细胞有丝分裂，在细胞凋亡和细胞周期的基因调控中发挥重要作用。正常情况下，survivin表达于胚胎和发育的胎儿组织细胞，不表达于终末分化的组织细胞。而在大多数人类肿瘤中，survivin在肿瘤细胞重新获得高表达，但在相应的癌旁组织中无表达。单核苷酸多态(singlenucleotide polymorphism，SNP)被认为是一种稳定遗传的早期突变，与疾病有着稳定的相关性。研究表明在survivin的启动子和编码区域均发现有SNPs变异，这些SNPs位点可能与survivin的表达调控相关，从而与机体对结直肠癌的易感性相关联。本研究探讨Survivin基因rs9904341C/G、rs8073069C/G单核苷酸多态性（SNP single nucleotide polymorphisms）与结直肠癌遗传易感性的相关性，为结直肠癌的诊治和预防提供有利的依据。方法：收集182例结直肠癌患者和200例健康者为研究对象，采用病例-对照研究方法，抽取外周静脉血各5ml，并记录个人有关资料。外周血DNA采用蛋白酶K消化-饱和氯化钠盐析法提取，采用聚合酶链反应-连接酶检测反应（polymerase chain reaction-ligase detection reaction，PCR-LDR）方法分别对rs9904341C/G、rs8073069C/G两个多态位点进行基因分型。采用SPSS13.0版软件包（SPSS Company, Chicago, Illinois, USA）进行数据统计分析，P＜0.05有统计学意义。对照组基因型频率分布用卡方检验进行Hardy-Weinberg平衡分析。病例组与正常对照组之间的基因型和等位基因型分布采用χ2检验进行比较。采用EH软件和2LD软件进行单体型频率和连锁不平衡分析。用非条件Logistic回归法计算表示相对风险度的比值比（odds ratio, OR）及95%可信区间（confidence interval, CI）。结果：1正常对照组中的rs9904341C/G的基因型分布频率符合Hardy-Weinberg平衡（P＞0.05），rs8073069C/G的基因型分布频率符合Hardy-Weinberg平衡（P＞0.05）。2Survivin基因rs9904341C/G多态的C、G等位基因频率在病例组和健康组中分别为59.3%、40.7%和49.7%、50.3%，两组相比认为有统计学意义（P=0.008, OR=1.474,95％CI=1.107~1.963）；在患者组和健康组中C/C、C/G、G/G基因型频率分别为：38.5%、41.7%、19.8%和26.5%、46.5%、27.0%，两组相比较有统计学意义（P=0.033）。CRC患者C/C基因型的频率（38.5%）明显高于对照人群（26.5%）。与C/C基因型相比，携带C/G和G/G基因型均可降低CRC的发病风险（OR=0.621,95％CI=0.389~0.992和OR=0.507,95％CI=0.292~0.882）。对个体吸烟人群行分层分析，发现在吸烟组中，与C/C基因型相比，携带C/G基因型可明显降低CRC的发病风险（OR=0.310,95％CI=0.119~0.808），携带G/G基因型与CRC的发病风险无相关性（OR=0.334,95％CI=0.103~1.083）。对个体饮酒人群进行分层分析，在饮酒组中，与C/C基因型相比，携带C/G基因型可显著降低CRC发病风险（OR=0.261,95％CI=0.090~0.754），携带G/G基因型与CRC的发病风险无相关性（OR=0.380,95％CI=0.103~1.407）。Survivin基因rs9904341C/G SNP对非吸烟人群和非饮酒人群CRC发病风险无影响（P＞0.05）。3Survivin基因rs8073069C/G的C、G等位基因频率在CRC组和健康组分别是30.5%、69.5%和27.7%、72.3%，两组相比较无统计学意义（P=0.404, OR=1.142，95％CI=0.836~1.561）；在病例组和对照组中C/C、C/G、G/G基因型频率分别为：11.0%、39.0%、50.0%和9.0%、37.5%、53.5%，两组相比无统计学意义（P＞0.05）。与C/C基因型比较，未发现携带C/G、G/G基因型对CRC发病风险的影响（OR=0.850，95％CI=0.420~1.740和OR=0.77，95％CI=0.380~1.530）。对个体吸烟、饮酒进行分层分析，发现与C/C基因型比较，携带C/G和G/G基因型对CRC的发病无影响。4采用EH软件和2LD软件对Survivin rs9904341与Survivin rs8073069两位点SNPs行联合分析，显示Survivin rs9904341与Survivinrs8073069两位点间存在连锁不平衡现象（D’=0.628）,最多见的单体型是rs9904341C-8073069G，在正常健康组中的频率为49.8%，其次为rs9904341G-8073069C（27.5%）、rs9904341G-8073069G（22.5%）和rs9904341C-8073069C（0.2%），人群中rs9904341C-8073069C单体型明显增加CRC发病风险（OR=54.86,95％CI=7.518~400.370）,与其单体型相比，rs9904341G-8073069C单体型可降低CRC发病风险（OR=0.595,95％CI=0.422~0.839）,其他两种单体型OR值分别为0.905（95％CI=0.681~1.202）、0.986（95％CI=0.701~1.386），此两种单体型可能与该病发病风险无关。结论：1本研究表明：Survivin基因rs9904341C/G SNP可能影响结直肠癌的发病风险，携带C/C基因型可明显增加结直肠癌的发病风险，与C/C基因型相比，携带C/G和G/G基因型可显著降低结直肠癌的发病风险。在吸烟和饮酒人群中，携带C/G基因型的患者与结直肠癌发病风险的降低有相关性。2Survivin基因rs8073069C/G SNP可能与结直肠癌的发病风险无关。3Survivin基因启动子区rs9904341C/G与rs8073069C/GSNPs位点间存在连锁不平衡（D’=0.628）,rs9904341C-8073069C单体型明显增加结直肠癌的发病风险,rs9904341G-8073069C单体型可降低结直肠癌发病风险,其他两种单体型可能与结直肠癌发病风险无关。更多还原

【Abstract】 Objective: Survivin is an inhibitor of apoptosis protein,which locksapoptosis induced by a variety of triggers.Survivin plays an important role incell cycle regulation，and may be involved in the development and progressionof cancers.The expression of survivin is ubiquitous in fetal issues,but isnegligible in the majority of terminally differentiated adult tissues. Single-nucleotide polymorphisms(SNP) are inheritable mutation and had steadyorrelation with various diseases.Several polymorphisms in survivin promoterhave been dentified previously.These SNPs might be correlated with the riskof tumors.The study was designed to investigate the correlation of Survivinrs9904341C/G、rs8073069C/GSNPs with the risk of CRC. By this way, wehope to offer some evidences for the prevention and therapy of CRC atmelocularbiological level.Methods: This population-based case-control study included182CRCpatients and200healthy controls.The genomic DNA was extracted by usingproteinase K digestion followed by a salting out procedure.Genotypes of theSurvivin gene were analyzed by polymerase chain reaction-ligase detectionreaction (PCR-LDR) method.Statistical analysis was performed using SPSS11.5software package. Aprobability level of5%was considered significant for all statistical analyses.Hardy-Weinberg analysis was performed by comparing the observed andexpected genotype frequencies in study groups using Chi-square test.Univariate comparisons of allele and genotype distribution in cases andcontrols were performed by means of two-sided contingency tables usingChi-square test.The Survivin haplotype frequencies and linkage disequilibriumcoefficient were estimated by using EH linkage software and2LD software.The odds ratio (OR) and95%confidence interval (CI) were calculated using an unconditional logistic regression model.Results：1The genotype frequencies of Survivin rs9904341C/G and rs8073069C/Gin healthy controls did not significantly deviate from that expected for aHardy-Weinberg equilibrium (P＞0.05).2The frequencies of the Survivin rs9904341C/G C and G allele among CRCpatients and healthy controls were59.3%、40.7%和49.7%、50.3%，respectively;There was significant difference between the two groups (P=0.008,OR=1.474,95％CI=1.107~1.963).The distribution of the C/C,C/G and G/G genotypesbetween CRC patients (38.5%、41.8%and19.8%, respectively) and controls(26.5%、46.5%and27.0%,respectively) had significant difference (P=0.033).The frequencies of the C/C genotype of the Survivin rs9904341C/G weresignificantly higher in CRC patients (38.5%) than those in healthy controls(26.5%).Compared with the C/C genotype,the C/G and G/G genotypes couldsignificantly reduce the risk of developing CRC,the odds ratio were0.621（95％CI=0.389~0.992）and0.507(95％CI=0.292~0.882).When stratified forsmoking status, the C/G genotype significantly reduced the risk of developingCRC among smoking patients compared with the C/C genotype with the ORof0.310(95％CI=0.119~0.808)respectively.No significant difference in theG/G genotype and the risk of developing CRC among smoking patientscompared with the C/C genotype with the OR of0.334(95％CI=0.103~1.083)respectively.When stratified for drinking status,the C/G genotype significantlyreduced the risk of developing CRC among drinking patients compared withthe C/C genotype with the OR of0.261(95％CI=0.090~0.754)respectively.Nosignificant difference in the G/G genotype and the risk of developing CRCamong drinking patients compared with the C/C genotype with the OR of0.380(95％CI=0.103~1.407)respectively.No significant difference in genotypeand allele distributions was found between patients and control groups inSurvivin rs9904341C/G (P>0.05),when stratified for non-smoking andnon-drinking status.3The frequencies of the Survivin rs8073069C/G C and G allele among CRC patients and healthy controls were30.5%,69.5%and27.8%,72.3%respectively; No significant difference in the Survivin rs8073069C/G alleledistribution was shown between CRC patients and controls (P=0.404).Thedistribution of the C/C、C/G and G/G genotypes between CRC patients (11.0%,39.0%and50.0%, respectively) and controls(9.0%,37.5%and53.5%,respectively) also had no significant differrence(P>0.05).Compared with theC/C genotype,the C/G and G/G genotypes could not increase the risk ofdeveloping CRC,the odds ratio were0.850(95％CI=0.420~1.740) and0.770（95％CI=0.380~1.530.No significant difference in genotype and alleledistributions was found between patients and control groups in Survivinrs8073069C/G (P>0.05),when stratified for smoking and drinking status.4The combined effect of Survivin rs9904341C/G and rs8073069C/G SNPswas analyzed by EH and2LD software.The results showed that Survivinrs9904341C/G and rs8073069C/G polymorphisms were link disequilibrium(D’=0.628).The result showed that the rs9904341C-8073069G haplotype wasthe most common, which was49.8%.The rs9904341C-8073069C haplotypeincrease the risk of CRC.Compared with rs9904341C-8073069C haplotype,The rs9904341G-8073069C haplotype reduce the risk of CRC.Two otherhaplotypes would not change onset risk of CRC.Conclusions:1The Survivin rs9904341C/G SNP was associated with the risk ofdeveloping CRC.The C/C genotype significantly increased the risk ofCRC,The C/G and G/G genotypes could significantly reduce the risk ofdeveloping CRC.The subjects, particularly smokers or drinkers,carryingC/G and G/G genotypes could be associated with the reduced risk ofdeveloping CRC.2The Survivin rs8073069C/G SNP might not be associated with the risk ofCRC development.3The Survivin rs9904341C/G and rs8073069C/G SNP was imperfectly inlinkage disequilibrium.The rs9904341C-8073069C haplotype increase therisk of CRC. Compared with rs9904341C-8073069C haplotype.The rs9904341G-8073069C haplotype reduce the risk of CRC.Two otherhaplotypes distribution had no influence on the risk of CRC.更多还原