【作者】 王雅锋；

【导师】 黄子扬；

【作者基本信息】 福建医科大学 ， 内科学（心血管内科）， 2017， 博士

【摘要】 第一部分MTHFR和SETD1A基因甲基化在慢性宫内缺氧对子代小鼠心肌结构影响中的作用研究背景:流行病学研究结果已显示胎儿所处的宫内不良环境与其成年期心血管疾病的发病相关,其机制尚未明确。缺氧是最重要的宫内不良环境之一。DNA甲基化与心血管疾病的发生发展密切相关。亚甲基四氢叶酸还原酶(methylenetetrahydrofolate reductase,MTHFR)和SET结构域蛋白1A(SET domain protein 1 A,SETD1A)分别是DNA甲基化和组蛋白甲基化过程中的关键酶。迄今为止,国内外尚无MTHFR和SETD1A基因甲基化与慢性宫内缺氧引起子代成年期小鼠心肌损伤关系的研究。目的:本研究旨在从DNA甲基化方面探讨MTHFR和SETD1A基因在慢性宫内缺氧导致子代小鼠心肌形态结构不良改变中的可能作用。方法:建立C57BL/6小鼠慢性宫内缺氧模型,培育3月龄及6月龄子代雄性小鼠,分为慢性宫内缺氧组(CIH组:CIH3、CIH6)和对照组(NC组:NC3、NC6),4组,每组10只,共40只,取左室心肌组织,观察心肌组织的形态学变化。测定MTHFR及SETD1A基因启动子区甲基化水平及其对两种基因mRNA及蛋白表达水平的影响。结果:心肌组织学分析显示CIH3、CIH6组心肌组织中总胶原、I型胶原含量、I/III型胶原的比值均较NC3、NC6组升高,凋亡的心肌细胞数量也较NC3、NC6组高;HE染色示CIH6组心肌组织出现排列不规则、细胞肿胀及溶解;CIH3、CIH6组中MTHFR基因启动子区甲基化水平分别较NC3、NC6组明显升高(P=0.025,P=0.010),MTHFR基因的mRNA(P<0.001)及蛋白表达水平(P=0.013,P=0.003)明显降低;SETD1A基因启动子区甲基化水平明显降低(P=0.032,P=0.048),SETD1A基因的mRNA(P<0.001,P=0.002)及蛋白表达水平(P=0.002,P<0.001)明显升高。结论:慢性宫内缺氧导致子代小鼠心肌纤维化、心肌细胞凋亡增加。慢性宫内缺氧诱发子代心肌结构改变可能与MTHFR和SETD1A基因甲基化水平改变进而改变其转录和翻译水平相关。第二部分MTHFR及SETD1A基因多态性与人类冠状动脉粥样硬化性心脏病风险的关系及其功能研究背景:冠心病是当今严重危害人类健康的常见病之一。单核苷酸多态性(single nucleotide polymorphisms,SNPs)是个体间外显性、疾病遗传易感性和药物反应性等个体生物学性状差异重要的遗传学基础。已有大量研究发现MTHFR基因rs1801133 C>T与冠心病的发生发展具有相关性。但是目前尚无关于MTHFR基因rs9651118 T>C、rs4846048 A>G、rs4845882 G>A、rs3753584 A>G和SETD1A基因rs9938550 G>A、rs1870293 C>T、rs2305881 C>T与冠心病发生风险关系的研究。目的:本研究旨在从DNA单核苷酸多态性方面探讨MTHFR和SETD1A基因与冠心病发生风险的关系及SNP对血浆MTHFR和SETD1A蛋白表达的影响。方法:选取MTHFR和SETD1A基因的8个标签位点,在中国东部地区冠心病病例组505例和正常对照组1109例中利用SNPscan对8个标签SNP位点进行基因分型,并进行回归分析计算风险比;同时,测定77个病例和93个对照组血液中MTHFR和SETD1A蛋白表达水平及不同基因型对蛋白表达的影响。结果:MTHFR rs1801133 CT和CT/TT基因型降低了冠心病的发生风险(CT vs.CC:adjusted OR=0.65,95%CI=0.51-0.83,P<0.001;CT/TT vs.CC:adjusted OR=0.70,95%CI=0.56-0.87,P=0.002),rs9651118 CC增加了冠心病的风险(CC vs.TT:adjusted OR=1.67,95%CI=1.21-2.31,P=0.002;CC vs.TT+TC:adjusted OR=1.91,95%CI=1.42-2.56,P<0.001);SETD1A rs2305881 CT基因型降低了冠心病的发生风险(CT vs.CC:adjusted OR=0.73,95%CI=0.54-1.00,P=0.047);在非心肌梗死的分层分析中也得到相似的结论。血浆中MTHFR和SETD1A蛋白含量分析表明,病例组中MTHFR蛋白含量较对照组明显减少(P<0.001),而SETD1A的含量较对照组升高(P<0.001),SETD1A rs2305881 CC基因型较CT基因型表达的蛋白水平升高(P=0.01),男性患者中MTHFR rs4846048 AG基因型表达的蛋白水平高于AA基因型(P=0.047),rs3753584 AG基因型表达的蛋白水平高于GG基因型(P=0.024),年龄<65岁的病人中,MTHFR rs4845882 AA基因型蛋白表达水平明显高于GG基因型(P=0.043),rs4846048 AG基因型蛋白表达水平明显高于AA基因型(P=0.012),rs4845882 AA基因型明显高于GG基因型的蛋白表达水平(P=0.043),不吸烟者MTHFR rs4846048 AG基因型蛋白表达水平明显高于AA基因型(P=0.032)。结论:MTHFR rs1801133 C>T和rs9651118 T>C与SETD1A rs2305881 C>T与冠心病的风险有关;冠心病病人血浆中所表达的MTHFR较对照组明显减少,而SETD1A较对照组明显增加;根据性别、年龄、吸烟情况对病例组MTHFR和SETD1A基因蛋白表达水平进行分层分析及组内比较,表明不同的人群特征SNP分布情况不同,其对蛋白表达水平的影响也不同。更多还原

【Abstract】 Part I: Effects of MTHFR and SETD1 A Gene Methylation on Myocardial Structure of Adult Mice Offspring suffering chronic intrauterine hypoxiaBackgrounds: The results of epidemiological studies have shown that the intrauterine environment in the fetus is associated with the onset of cardiovascular disease in adult life,and its mechanism is not clear.Hypoxia is one of the most important intrauterine environments.DNA methylation is closely related to the development of cardiovascular disease.Methylenetetrahydrofolate reductase(MTHFR)and SET domain protein 1 A(SETD1A)are the key enzymes in the process of DNA methylation and histone methylation,respectively.So far,there is no study on the relationship between MTHFR and SETD1 A gene methylation and myocardial damages caused by chronic intrauterine hypoxia(CIH)in adult mice offspring.Objective: The aim of the present study was to investigate whether the structural changes of mice offspring heart caused by chronic intrauterine hypoxia may relate to the promoter methylation of MTHFR and SETD1 A gene.Methods: We established chronic intrauterine hypoxia model of C57BL/6 mouse.Three and six months old offspring of male mice were cultivating and divided into chronic intrauterine hypoxia group(CIH group: CIH3,CIH6)and control group(NC group: NC3,NC6),with a total of 24 mice,4 groups,6 mice in each group.The left ventricular myocardium of each mouse was selectd.MTHFR and SETD1 A gene promoter methylation levels and their effects on gene expression were determined.And the morphologic changes of myocardium were observed.Results: Myocardial histological analyses showed that the contents of total collagen and collagen type I and the ratio of collagen type I/III in CIH3 and CIH6 groups were higher than those in group NC3 and NC6.The number of apoptotic myocardial cells were higher than that of NC3 and NC6 group.HE staining demonstrated that the myocardial tissue of CIH6 group showed irregular arrangement,cell swelling and lysis.Compared to NC3 and NC6,the methylation level of MTHFR gene promoter in CIH3 and CIH6 was significantly increased(P = 0.025,P = 0.010,respectively)and mRNA(P < 0.001,P < 0.001,respectively)and protein expression levels(P = 0.013,P = 0.003,respectively)were significantly decreased;the methylation level of SETD1 A gene promoter was significantly decreased(P = 0.032,P = 0.048,respectively)and the mRNA(P < 0.001,P = 0.002,respectively)and protein expression levels(P = 0.002,P < 0.001,respectively)were significantly increased.Conclusions: Chronic intrauterine hypoxia leads to increased myocardial fibrosis and myocardial cell apoptosis in offspring mice.As a predisposing factor for the changes of myocardial structure in offspring mice,myocardial lesions induced by chronic intrauterine hypoxia may be related to changes of MTHFR and SETD1 A gene methylation levels and their transcription and translation levels successively.Part II: Relationship between MTHFR and SETD1 A Polymorphisms and Their functions and Coronary Heart Disease Risk in Human Backgrounds: Coronary heart disease(CHD)is one of the common diseases that seriously endangers human health today.Single nucleotide polymorphisms(SNPs)are important genetic basis for the differences of individual biological traits such as interindividual dominance,genetic susceptibility of diseases and drug reactivity.There has been a great deal of researches to find that MTHFR rs1801133 C>T has a correlation with the risk of CHD.However,there is no research on the relationship between MTHFR rs9651118 T> C,rs4846048 A> G,rs4845882 G> A,rs3753584 A>G and SETD1 A rs9938550 G> A,rs1870293 C> T,rs2305881 C>T and CHD risk.Objective: The aim of the present study was to investigate the association between MTHFR and SETD1 A single nucleotide polymorphisms(SNPs)and coronary heart disease(CHD)risk and the effects of SNPs on protein expression level.Methods:We selected eight MTHFR and SETD1 A gene tagging SNPs.SNPscan was used to analyze the genotyping of eight SNPs in five hundred and five cases of coronary heart disease and 1109 cases of normal controls from Eastern China.And the risk ratios were calculated by regression analysis.Results:MTHFR rs1801133 CT and CT/TT genotypes decreased CHD risk(CT vs.CC: adjusted OR = 0.65,95%CI = 0.51-0.83,P < 0.001;CT + TT vs.CC: adjusted OR = 0.70,95%CI = 0.56-0.87,P = 0.002),rs9651118 CC increased CHD risk(CC vs.TT: adjusted OR = 1.67,95%CI = 1.21-2.31,P = 0.002;CC vs.TT + TC:adjusted OR = 1.91,95%CI = 1.42-2.56,P < 0.001);SETD1A rs2305881 CT decr eased CHD risk(CT vs.CC: adjusted OR = 0.73,95%CI = 0.54-1.00,P = 0.047).The content of MTHFR protein in the cases was significantly lower than that of controls group(P < 0.001).The content of SETD1 A in the cases was higher than that of controls group(P < 0.001).The protein level of SETD1 A rs2305881 CC genotype was higher than that of CT genotype(P = 0.01).In male patients,the protein level of MTHFR rs4846048 AG genotype was higher than that in AA genotype(P = 0.047)and the protein level of rs3753584 AG genotype was higher than that of GG genotype(P = 0.024).In the cases aged <65 years,the protein level of MTHFR rs4845882 AA genotype was significantly higher than that of GG genotype(P = 0.043),that of rs4846048 AG genotype significantly higher than AA genotype(P = 0.012)and that of rs4845882 AA genotype significantly higher than GG genotype(P = 0.043).The protein level of MTHFR rs4846048 AG was significantly higher in non-smokers than in AA genotype(P = 0.032).Conclusions: MTHFR rs1801133 C>T and rs9651118 T>C and SETD1 A rs2305881 C>T were associated with the risk of CHD.Stratified by gender,age and smoking,the distribution of SNPs genotyping were different and its influences on protein expression were also different.更多还原