【作者】 黄根；

【导师】 张焜和；

【作者基本信息】 南昌大学 ， 内科学， 2009， 硕士

【摘要】 背景与目的:乙肝病毒感染后有不同的临床转归,其中肝硬化危害最大。乙肝后肝硬化的发生不仅与环境因素和病毒因素有关,还与个体的遗传易感性密切相关。单核苷酸多态性(SNP)占人类可遗传变异的90%以上,是目前诠释疾病遗传易感性最好的遗传标记。细胞因子基因多态性与乙肝后肝硬化的关系已有较多的研究报道,但与肝硬化发生过程有关的一些非免疫基因多态性很少或没有报道,更缺乏较系统的研究。本项目将联合检测7个与肝硬化发生过程相关的非免疫基因多态性位点,探讨它们与乙肝后肝硬化遗传易感性的关系。方法:(1)研究对象:乙肝后肝硬化168例(病例组)和无症状乙肝病毒携带者155例(对照组)。(2)基因多态性分析:酚-氯仿法提取外周血基因组DNA,采用限制性片段长度多态性(PCR-RFLP)方法分析各基因位点的基因型。(3)计算各位点的基因型和等位基因频率,采用卡方检验比较它们的组间差异,并进行Hardy-Weinberg平衡吻合度检验。(4)分别以总样本和性别分层样本进行单因素Logistic回归分析,计算各基因型及等位基因的比数比(OR)及95%可信区间,并进行多因素Logistic回归分析筛选独立的危险和保护基因型和等位基因。结果:(1)血管紧张素原基因-20A/C多态位点的各基因型及等位基因频率在肝硬化组和HBsAg携带组之间差异无统计学意义(P>0.10),但多因素分析显示与女性HBV感染者肝硬化风险可能有一定关系,AA基因型和C等位基因的OR接近有统计学意义(P=0.064、0.080)。(2)血管紧张素原基因-6A/G多态位点的基因型及等位基因频率在肝硬化组和HBsAg携带组之间差异无统计学意义(P>0.10),但多因素分析显示男性HBV感染者AA是易感基因型(OR=1.927, P=0.047),G是保护性等位基因(OR=0.508, P=0.047)。(3)雌激素受体α基因-29T/C多态位点的各基因型和等位基因频率在肝硬化组和HBsAg携带组之间差异无统计学意义(P>0.10),但单因素Logistic回归分析显示该位点与女性乙肝后肝硬化易感性关系密切,TC为易感基因型(OR=3.336, P=0.007),CC为保护基因型(OR=0.327, P=0.015),T为易感等位基因(OR=3.061, P=0.015),多因素分析仍显示TC是女性易感基因型(OR=3.454,P=0.027),T是易感等位基因(OR=3.554,P=0.024)。(4)基质金属蛋白酶9基因-1562C/T位点的各基因型和等位基因频率在肝硬化组和HBsAg携带组之间都非常接近,无统计学意义(P>0.10),单因素和多因素分析也未显示其与肝硬化易感性有关。(5)微粒体环氧化物水解酶基因Tyr113His位点的各基因型和等位基因频率在肝硬化组和HBsAg携带组之间差异无统计学意义(P>0.10),单因素和多因素分析也未显示它们与乙肝后肝硬化易感性有关。(6)微粒体环氧化物水解酶基因-613C/T位点的各基因型和等位基因频率在肝硬化组和HBsAg携带组之间差异也无统计学意义(P>0.10),但该位点与女性乙肝后肝硬化易感性可能有一定的关系,单因素分析显示TT基因型和C等位基因的OR接近有统计学意义(P=0.089)。(7)谷胱甘肽转硫酶M1有空白基因型和正常基因型两种,其各基因型频率在肝硬化组和HBsAg携带组之间都非常接近,无统计学意义(P>0.10),多因素分析也未显示其与乙肝后肝硬化相关。(8)多因素Logistic回归分析显示本研究所检测的7个基因多态性位点中,AGT-6AA是独立的男性乙肝后肝硬化易感基因型,G是独立的男性保护性等位基因,ESR1+29TC基因型和T等位基因都是独立的女性乙肝后肝硬化的危险因子。结论:(1)血管紧张素原基因-20A/C和-6A/G位点多态性与乙肝后肝硬化易感性有一定关系,其中-6A/G位点与男性乙肝后肝硬化易感性相关,-20A/C位点与女性乙肝后肝硬化易感性有一定相关性。(2)ESR1基因+29T/C位点基因多态性与女性乙肝后肝硬化易感性密切相关,与男性无明显相关。(3)基质金属蛋白酶9基因-1562C/T位点与乙肝后肝硬化易感性无明显相关。(4)微粒体环氧化物水解酶基因-613C/T和Tyr113His两个多态位点均与乙肝后肝硬化易感性无明显相关。(5)GSTM1空白或正常基因型与乙肝后肝硬化易感性无明显相关。(6)AGT-6A/G位点是与男性乙肝后肝硬化易感性相关的独立因素,ESR1 +29T/C位点是与女性乙肝后肝硬化易感性相关的独立因素。更多还原

【Abstract】 Background and Objective: There are different clinical outcomes in patients with hepatitis B virus infection, and the most serious condition is hepatic cirrhosis. The development of HBV-related hepatic cirrhosis is associated with not only environmental and viral factors but also hereditary susceptibility. Single nucleotide polymorphisms (SNPs), account for more than 90% of human genetic variations, are the best genetic markers to indicate genetic susceptibility of diseases. There are some studies reported on the relationships between cytokine gene polymorphisms and the HBV-related hepatic cirrhosis, but few studies on the non-immune-related gene polymorphisms associated with the development of HBV-related cirrhosis, and lack of systematic researches about them. In the present study, we detected 7 genetic polymorphism sites of non-immune-related genes possiblely associated with the development of liver cirrhosis, and analyzed the relationship between them and the susceptibility of HBV-related hepatic cirrhosis.Methods: (1) Patients: 168 cirrhosis patients with HBV infection (case group) and 155 asymptomatic HBV carriers (control group) were recruited. (2) Gene polymorphism analysis: peripheral blood genomic DNA was extracted by phenol-chloroform method. Polymerase chain reaction-restriction fragment length polymorphism was used to determine the genotypes of each polymorphism sites. (3) Genotype and allele frequencies in each site were calculated and their differences between two groups were compared by chi-square test, and Hardy-Weinberg equilibriums of genotypes was verified by chi-square test. (4) The odds ratios (ORs) and their 95% confidence intervals of all genotypes and alleles were calculated by univariate Logistic regression analysis in total sample and gender-stratified sub-samples, respectively. Multivariate Logistic regression analysis was used to screen independent risk and protective genotypes and alleles.Results: (1) The differences of all genotype and allele frequencies at AGT-20A/C site were not significant between case group and control group (P>0.10). However, multivariate analysis showed a probable association between this site and susceptibility of HBV-related cirrhosis in female patients, the ORs of genotype AA and allele C nearly significant (P=0.064, 0.080). (2) The differences of all genotype and allele frequencies at AGT -6A/G site were not significant between case group and control group (P>0.10), but multivariate analysis showed that AA was a risk genotype (OR=1.927, P=0.047) and G was protective allele (OR=0.508, P=0.047) in male patients. (3) The differences of all genotype and allele frequencies at ESR1 +29T/C site were not significant between case group and control group (P>0.10). However, univariate analysis showed that it is closely associated with susceptibility of female cirrhosis. The TC was susceptible genotype (OR=3.336, P=0.007), CC was a protective genotype (OR=0.327, P=0.015), and T was susceptible allele (OR=3.061, P=0.015) in female patients. Multivariate analysis confirmed that TC was susceptible genotype (OR=3.454,P=0.027) and T was susceptible allele (OR=3.554,P=0.024) in female. (4) The differences of all genotype and allele frequencies at MMP9 -1562C/T site were not significant between case group and control group (P>0.10), and univariate and multivariate analyses did not show association between it and susceptibility of HBV-related cirrhosis. (5) The differences of all genotype and allele frequencies at MEH Tyr113His site were not significant between case group and control group (P>0.10), and univariate and multivariate analyses did not show association between it and susceptibility of HBV-related cirrhosis. (6) The differences of all genotype and allele frequencies at MEH -613C/T site were not significant between case group and control group (P>0.10), but there seemed some correlation between this site and susceptibility of female HBV-related liver cirrhosis. In female patients, the ORs of TT genotype and C allele were nearly significant (P=0.089, 0.089). (7) The differences of GSTM1 genotype frequencies were not significant between case group and control group (P>0.10), and univariate and multivariate analyses did not show that this site is associated with susceptibility of HBV-related liver cirrhosis. (8) Multivariate Logistic regression analysis showed that in the 7 genetic variations detected, AGT-6AA was an independent risk genotype and AGT-6G was an independent protective allele for HBV-related cirrhosis development in male patients, and both of ESR1+29TC genotype and T allele were risk factors for HBV-related cirrhosis development in female patients.Conclusions: (1) AGT gene polymorphisms are associated with susceptibility of HBV-related liver cirrhosis, in which -6A/G site correlated with susceptibility of male cirrhosis, and -20A/C correlated with susceptibility of female cirrhosis. (2) There is significant association between gene polymorphisms at position +29T/C of ER1 and susceptibility of female HBV-related liver cirrhosis, but without significant association with male. (3) There is no significant association between gene polymorphisms at position -1562 of MMP-9 and susceptibility of HBV-related liver cirrhosis. (4) There is no significant association between gene polymorphisms at position -613C/T or -Tyr113His of MEH and susceptibility of HBV-related liver cirrhosis. (5) There is no significant association between null or normal genotype of GSTM1 and susceptibility of HBV-related liver. (6) AGT-6A/G site is an independent factor for male cirrhosis development and ESR1+29T/C site is an independent factor for female cirrhosis development.更多还原