【作者】 高会影；

【导师】 李利；

【作者基本信息】 河北医科大学 ， 妇产科学， 2010， 硕士

【摘要】 目的:醌氧化还原酶1(NQO1)是真核细胞内普遍存在的一类黄素蛋白酶,定位于人类染色体16p22。该酶催化醌双电子还原反应,对醌及其衍生物有解毒作用,能够解除醌类物质对细胞的毒害,从而起到保护细胞的作用,是人体内一种重要的化学致癌物代谢酶。NQO1第六位外显子(C609T),第四位外显子(C465T)为非同义编码单核苷酸多态(ns-cSNP),可引起蛋白质编码氨基酸序列改变从而导致蛋白质功能的改变,进而影响肿瘤的发生发展。因此,雌激素代谢过程中NQO1酶的活性可能与子宫内膜癌发生有密切关系。本研究旨在探讨NQO1单核苷酸多态性与中国北方妇女子宫内膜癌发病风险的关系。方法:本研究采用病例-对照研究方法,209例病例来源于河北医科大学第四医院妇产科住院手术治疗的子宫内膜癌患者,所有病例均经病理学确诊。209例健康对照来自同期在同一医院进行健康体检的无关个体。均采集外周静脉血5ml,采用蛋白酶K消化-饱和氯化钠盐析法提取外周血白细胞DNA。采用蛋白酶K消化-饱和氯化钠盐析法提取外周血白细胞DNA,采用聚合酶链反应-限制性片段长度多态性(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)分析方法检测NQO1 C609T、C465T基因多态位点的基因型频率分布。数据统计分析采用SPSS11.5版软件包(SPSS Company,Chicago,Illinois,USA)进行,P<0.05认为有统计学意义。对照组基因型频率进行Hardy-Weinberg平衡分析。病例组与对照组的基因型、等位基因型分布比较均采用行×列表卡方检验。以非条件Logistic回归法计算相对风险度的比值比(odds ratio,OR)及其95%可信区间(confidence interval,CI)。结果:1.初潮年龄及是否绝经与子宫内膜癌发病风险的关联分析1.1初潮年龄在病例组和对照组的平均值分别为15.48、15.03岁,病例组初潮年龄≥15岁比例(47.4%)明显高于对照组(36.8%)。经统计分析发现,初潮年龄≥15岁的个体其子宫内膜癌的患病风险是初潮年龄<15岁个体的1.543倍,说明初潮年龄晚可能是子宫内膜癌的易感因素。1.2根据是否绝经将病例组和对照组分为已绝经组和未绝经组,病例组所占频率分别为67.9%和32.1%,对照组为61.7%和38.3%。两组比较结果无显著性差异(P>0.05)。2. NQO1 C609T与子宫内膜癌的关系2.1统计学处理显示对照组的基因型频率分布均符合Hardy-Weinberg平衡(X2=0.445,P=0.505)。NQO1 C609T SNP的3种基因型频率(野生型C/C、杂交型C/T、突变型T/T)在病例组和对照组中分别是15.8%、65.1%、19.1%和29.7%、51.7%、18.7%,两组比较有显著性差异(X2=12,078,P=0,002)(Table 3)。病例组的T等位基因频率(51.7%)明显高于对照组(44.5%),两组比较有统计学意义(X2=4.313,P=0.038)。与C/C基因型比较,携带C/T+T/T基因型显著增加子宫内膜癌的发病风险(OR=2.249,95% CI=1.398-3.620)。2.2根据初潮年龄进行分层分析,发现NQO1 C609T多态基因型在初潮年龄<15岁的病例组中的分布与对照组相比具有统计学意义(P<0.05),而初潮年龄≥15岁无统计学意义。C/C, T/T和C/T基因型在初潮年龄<15岁病例组中的分布与对照组相比,差异有统计学意义(P=0.001,OR=3.089,95%CI=1.579~6.044)。结果表明与C/C基因型相比,携带T/T和C/T基因增加初潮年龄<15岁个体患子宫内膜癌的发病风险。2.3根据是否绝经进行分层分析,发现NQO1 C609T多态基因型在未绝经病例组的分布与对照组相比均有统计学意义(P<0.05)而在已绝经组无统计学意义。与C/C基因型比较,携带T/T和C/T的基因型在未绝经病例组的分布与对照组相比,病例组中基因型频率明显高于对照组,两组比较有统计学意义(P=0.007,OR=3.757,95%CI=1.571~8.984)。结果表明与C/C基因型相比,携带T/T和C/T基因增加未绝经个体患子宫内膜癌的发病风险。3. NQO1 C465T SNPs与子宫内膜癌的关系3.1统计学处理显示对照组NQO1 C465T SNPs的基因型频率分布均符合Hardy-Weinberg平衡(X2=0.14,P=0.71)。NQO1 C465T SNP的3种基因型频率(野生型C/C、杂交型C/T、突变型T/T )在病例组和对照组中分别是49.3%、46.9%、3.8%和55.5%、37.3%、7.2%,两组比较无显著性差异(X 2=1.661,P=0.436)。与C/C基因型比较,携带C/T+T/T基因型尚不能增加子宫内膜癌的发病风险(OR=1.284 , 95% CI=0.874-1.866)。3.2根据初潮年龄进行分层分析,发现NQO1 C465T多态基因型在初潮年龄<15岁和初潮年龄≥15岁的病例组与对照组相比均无统计学意义(P>0.05)。与C/C比较,T/T和C/T基因型在初潮年龄<15岁和初潮年龄≥15岁的病例组与对照组中,两组比较均无统计学意义。3.3根据是否绝经进行分层分析,发现NQO1 C465T多态基因型在未绝经和已绝经的病例组的分布与对照组相比均无统计学意义(P>0.05)。与C/C基因型比较,T/T和C/T基因型在初潮年龄<15岁和初潮年龄≥15岁的病例组与对照组中,两组比较均无统计学意义。4. NQO1 C609T与NQO1 C465T连锁不平衡分析采用2LD软件分析发现NQO1 C609T和NQO1 C465T SNPs间不存在连锁不平衡关系(D’=0.117157)。结论:1初潮年龄≥15岁的个体其子宫内膜癌的患病风险是初潮年龄<15岁个体的1.543倍,表明初潮年龄晚可能是子宫内膜癌的易感因素之一。2 NQO1C609T多态性中,C/T、T/T、C/T+T/T基因型均显著增加子宫内膜癌的发病风险。根据是否绝经和初潮年龄分层分析发现,携带C/T、T/T基因型可增加未绝经和初潮年龄早的个体子宫内膜癌的患病风险。3 NQO1C465T基因多态中突变的基因型及等位基因型与子宫内膜癌的发病风险无相关性。根据是否绝经和初潮年龄分层分析也未发现明显关联。4 NQO1C465T和NQO1C609T基因多态性之间联合突变在子宫内膜癌发病风险中不具有协同作用。更多还原

【Abstract】 Objective: Quinone oxidoreductase 1 (NQO1) is a kind of flavoprotein enzyme existing in the eukaryotic cell, which locate at the humankind chromosome 16p22. It is an important chemical carcinogen metabolic enzyme, which catalyzes quinone-electron reduction reaction and protects the cells from toxic effect of quinone and it’s derivate, by reducing their effects. Exons(C609T)and(C465T)are non-synonymous coding mononucleotide polymorphism, which may cause the change of protein coding amino acid sequence and the change of the protein function, then influence the occurrence and development of tumor. Therefore, the activity of NQO1 enzyme may have a close relationship with the occurrence of endometrial cancer in the process of estrogen metabolism. The research is to explore the relationship of NQO1 mononucleotide polymorphism with the risk of endometrial cancer in northern chinese women.Methods: The population-based case-control study included 209 endometrial cancer patients, who were done operations in the fourth hospital of Hebei Medical University, and were proven by pathology. And 209 healthy controls were from the same hospital for physical examination. Genomic DNA was extracted by using proteinase K digestion followed by a salting out procedure. Polymorphisms of NQO1 C609 T, C465T gene were analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis.Statistical analysis was performed using SPSS11.5 software package. P<0.05 was considered significant. Hardy-Weinberg analysis was performed by comparing the observed and expected genotype frequencies in the control group using Chi-square test. Comparison of the C609T and C465T genotypes and allelotypes distributions in cancer patients and healthy controls were performed by means of two-sided contingency tables using Chi-square test. The odds ratio (OR) and 95% confidence interval (CI) were calculated using an unconditional logistic regression model.Results:1 The correlation of menarche age and menopause with the risk of endometrial cancer1.1 The menarche age is 15. 48 years old in case, and 15.03 in control group. The percentage of women whose menarche age were older than 15 in case, was obviously greater than that in control. By statistical analysis, the individuals with menarche age≥15 had 1.543 times risk to get endometrial cancer than those under 15, indicating the older menarche age is one of the predisposing factor of endometrial cancer.1.2 According to menopause condition, the case group and control group were divided into pre-menopause and post-menopause group. The percentage of two groups were 32.1% and 67.9% in case, 38.3% and 61.7% in control. The difference was not significant between case and control groups (P>0.05).2 Gene NQO1 C609T polymorphism with the risk of endometrial cancer2.1 The distributions of NQO1 C609T genotypes among healthy controls did not significantly deviate from that expected by Hardy-Weinberg equilibrium (X2= 0.445, P= 0.505). The frequency of wild genotype (C/C), heterozygous genotype (C/T), and homozygous genotype (T/T) were 15.8%, 65.1%, 19.1% in case, 29.7%, 51.7%, 18.7% in control. The frequency of the T allele in patients (51.7%) was significantly higher than that in the controls (44.5%) (X2=4.313, P=0.038). Compared with C/C the genotype, merging T/T and C/T genotype, significantly increased the risk of endometrial cancer ( OR= 2.249). 2.2 According to menarche age, there was significant difference between the distribution of NQO1 C609T gene polymorphism in case group and that in control group while menarch age <15 (P<0.05). But there was no significant difference while menarche age≥15. The genotype distribution of C/C, C/T and T/T in case group was significantly different from that in control group while menarch age <15 (P=0.001, OR=3.089, 95%CI =1.579~6.044). Contrast with C/C genotype, merging T/T and C/T genotypes, the risk of developing endometrial cancer was increased while menarch age <15 (P=0.001, OR=3.089, 95%CI=1.579~6.044).2.3 According to whether or not menopause, the gene polymorphisms of NQO1 C609T were different between the case and control group while premenopause (P<0.05), but no difference while post-menopause (P>0.05). Contrast with C/C the genotype, merging T/T and C/T genotypes, the risk of developing endometrial cancer was increased while premenopause (P=0.007, OR=3.757, 95%CI=1.571~8.984).3 Gene NQO1 C465T polymorphism with the risk of endometrial cancer3.1 The distributions of NQO1 C465T genotypes among healthy controls did not significantly deviate from that expected by Hardy-Weinberg equilibrium (X2=0.14,P=0.71). The frequency of C/C, C/T and T/T genotypes were 49.3%、46.9%、3.8% in case and 55.5%、37.3%、7.2% in control. The frequency of the T allele in patients (27.3%) was not significantly different from that in the controls (25.8%) (X2=4.313, P=0.038). No significant difference was found either when the distribution of C/C genotype was compared with the T/T+C/T genotypes (P=0.436, OR= 1.284, 95% CI =0.874-1.866).3.2 According to menarche age, there was no significant difference between the case and control group while menarche age <15 (P>0.05), and either while menarche age≥15. No significant difference was found either when the distribution of C/C genotype was compared with the T/T+C/T genotype (P=0.436, OR= 1.284, 95% CI=0.874-1.866).3.3 According to whether or not menopause, the polymorphisms of NQO1 C465T were not different between the case and control group while premenopause (P>0.05), and while post-menopause either (P>0.05). No significant difference was found either when the distribution of C/C genotype was compared with the T/T+C/T genotypes (P=0.436, OR=1.284, 95% CI=0.874-1.866).4. Linkage disequilibrium analysis of NQO1 C609T and NQO1 C465TThere was no unification in genetic polymorphism of NQO1 C609T and NQO1 C465T on the risk of endometrial cancer by 2LD software.Conclusions:1 The individual with menarche age≥15 has 1.543 times of risk to get endometrial cancer, indicating the older menarche age is one of the predisposing factors in endometrial cancer.2 Among the NQO1 C609T gene polymorphism, the C/C and T/T genotypes increase the risk of endometrial cancer. Stratified by menarch age and menopause condition, the individual with C/C or T/T genotypes is susceptible to the endometrial cancer while premenopause or early menarche age.3 The NQO1 C465T polymorphism has no association with the risk of endometrial cancer. According to menarche age and menopause condition, there were no relationships with the endometrial cancer as well.4 There was no unification between NQO1 C609T gene polymorphism and NQO1 C465T on the risk of endometrial cancer.更多还原