外周血白细胞DNA甲基化调控在冠心病发病机制中的作用研究

发布时间：2018-03-04 15:13

本文选题：冠心病　切入点：色谱-质谱联用　出处：《武汉大学》2015年博士论文　论文类型：学位论文

【摘要】：冠心病(Coronary Artery Disease,CAD)是一类由遗传,环境及其之间的相互作用引起的复杂疾病,确切致病原因至今尚未阐明。多年来流行病学的研究表明,除了遗传因素以外,生活方式,饮食习惯,环境暴露等均与冠心病的发病风险相关。DNA甲基化是一种发生在DNA序列上的表观遗传修饰,可以在转录和细胞分裂前后稳定的遗传。越来越多的报道证实,高脂血症、吸烟、不良的生活习惯等心血管危险因素均可导致相关基因甲基化异常,从而促进心血管疾病的发生发展。由于研究人群的不一致及方法学的局限,以往的研究结果对于冠心病患者外周血白细胞DNA总体甲基化水平的变化情况没有取得一致的认识。本课题研究第一部分,共收集外周血样本435例(215例冠心病患者,220例健康对照),利用色谱-质谱联用技术检测外周血白细胞DNA总体甲基化(5-mdC/dG,m6A/A)水平,并用荧光定量PCR技术检测了3个DNA甲基化转移酶(DNM1,DNMT3A,DNMT3B)及3个脂代谢相关基因(SREBP-1,HMGCR, MLXIP)mRNA的表达水平,通过统计学检验两组样本之间甲基化水平及基因表达水平的差异。第二部分,建立THP-1泡沫细胞模型,利用色谱-质谱联用技术检测单核细胞泡沫化前后DNA总体甲基化水平的改变情况；在THP-1泡沫细胞中转染DNMT1全长质粒,采用质谱法检测SREBP-1启动子区域甲基化水平的改变情况,并通过荧光定量PCR,Western Blot技术检测脂代谢基因SREBP-1 mRNA及蛋白表达情况。第三部分,共收集外周血样本954例(476例冠心病患者和478例健康对照),依据Haploview软件提供的SNP连锁图挑取5个DNMT1 tag-SNP位点(rs2228611, rs16999593, rs7253062,rs2336691, rs4804494)。采用高分辨率熔解曲线法(HRM)检测样本中此5个SNP位点基因型,并通过统计学检验分析两组人群中5个位点及单体型的分布差异情况。该课题第一部分主要研究发现：1)研究中健康汉族人群外周血白细胞样本DNA总体5-mdC/dG水平和年龄,外周血清总胆固醇水平呈负相关；2)冠心病患者外周血白细胞基因组5-mdC/dG含量显著低于对照人群,总体5-mdC/dG含量降低是冠心病的潜在风险因素(odds ratio(OR)=2.454,95% confidence interval(CI)=1.665～3.617,校正P=5.789×10-6)；患者外周血白细胞基因组m6A/A含量显著高于对照人群,m6A/A含量增高是冠心病的潜在风险因素(OR=0.655,95%CI=0.448～0.957,校正P=0.029)；3)冠心病患者外周血白细胞DNMT1 mRNA表达水平显著增高,SREBP-1 mRNA表达水平显著降低。4)THP-1单核细胞泡沫化进程中基因组总体甲基化水平(5-mdC/dG, m6A/A)显著降低,DNMT1表达水平降低；5)THP-1泡沫细胞转染DNMT1全长质粒后,SREBP-1启动子特定CG位点启动子甲基化水平增高,SREBP-1 mRNA及蛋白表达水平降低。6)DNMT1基因上两个多态性位点(rs2228611和rs2336691)的基因型与CAD的患病风险有关联；7)rs7253062与脂质代谢相关；8)在单体型分析中基因型G-T-A-A-T(等位基因顺序依次为：rs2228611, rs16999593, rs7253062, rs2336691和rs4804494)与CAD的患病风险相关联。本研究提示DNA甲基化调控在冠心病的发病机制中扮演了重要角色,外周血白细胞DNA整体甲基化水平减低可能是冠心病的潜在风险因素；冠心病病理进程中的重要步骤-单核细胞的泡沫化受到了甲基化的调控,脂质代谢基因SREBP-1启动子区异常的甲基化改变了该基因的正常表达；DNMT1的基因多态性与冠心病关联。
[Abstract]:Coronary heart disease (Coronary Artery Disease, CAD) is a complex disease caused by genetic and environmental interaction between and the exact cause has not been elucidated. Years of research and Epidemiology showed that besides genetic factors, lifestyle, dietary habits, environmental exposure and incidence of coronary artery disease were risk associated with.DNA methylation is epigenetic modifications occur in an DNA sequence on the table, in the genetic transcription and cell division before and after stable. More and more reports confirmed, hyperlipidemia, smoking, bad habits and other cardiovascular risk factors can lead to abnormal methylation of related genes, so as to promote the development of cardiovascular disease. Because the study group the inconsistencies and methodological limitations of previous research results of DNA for white blood cells in peripheral blood of patients with coronary heart disease total methylation level did not have a By understanding this topic. The first part of the study, collected peripheral blood samples of 435 cases (215 cases of coronary heart disease patients and 220 healthy controls), using gas chromatography-mass spectrometry DNA white blood cell detection in peripheral blood total methylation (5-mdC/dG, m6A/A) level, using fluorescence quantitative PCR detection of 3 DNA methyltransferase (DNM1, DNMT3A, DNMT3B) and 3 genes related to lipid metabolism (SREBP-1, HMGCR, MLXIP) the expression level of mRNA, test the expression of methylation level and gene between the two sample groups by statistics. In the second part, the establishment of THP-1 foam cell model, combined with the changes of detection before and after bubble monocyte DNA total methylation level by gas chromatography - mass spectrometry; in THP-1 foam cells stained transfer DNMT1 plasmid, the change detection of SREBP-1 promoter methylation level by mass spectrometry, and by fluorescence quantitative PCR, Western The expression of Blot in detection of lipid metabolism gene SREBP-1 mRNA and protein. The third part, collected peripheral blood samples of 954 cases (476 cases of coronary heart disease patients and 478 healthy controls), 5 DNMT1 tag-SNP sites based on Haploview software provided by the SNP linkage map (rs2228611, rs16999593, were rs7253062, rs2336691, rs4804494) with high. Resolutionmelting curve method (HRM) detection in a sample of the 5 SNP loci, and through statistical analysis of differences between the two groups in the distribution of 5 loci and haplotypes. The first part mainly study found that: 1) the healthy Han population of peripheral white blood cell samples DNA 5-mdC/dG level and overall age was negatively related to total cholesterol levels in peripheral blood of patients with coronary heart disease; 2) peripheral white blood cell genomic 5-mdC/dG content was significantly lower than the control group, the overall 5-mdC/dG content reduced latent coronary heart disease In the risk factors (odds ratio (OR) =2.454,95% confidence interval (CI) =1.665 ~ 3.617, corrected P=5.789 x 10-6); patients with peripheral white blood cell genomic m6A/A content was significantly higher than that of the control group, the increase of m6A/A content is a potential risk factor for coronary heart disease (OR=0.655,95%CI= 0.448 ~ 0.957, P=0.029; 3) correction) patients with coronary heart diseases white blood cells DNMT1 mRNA expression level was significantly increased, the expression level of SREBP-1 mRNA decreased significantly.4) level in whole genome methylation of THP-1 foam mononuclear cells in the process (5-mdC/dG, m6A/A) significantly decreased, the expression level of DNMT1 decreased; 5) THP-1 foam cells transfected with DNMT1 full-length plasmid, SREBP-1 promoter methylation of specific CG sites the level of increased expression of SREBP-1 protein and mRNA levels decreased.6) DNMT1 gene two polymorphism loci (rs2228611 and rs2336691) associated risk genotype and the prevalence of CAD Rs7253062; 7) and associated with lipid metabolism; 8) in haplotype analysis in genotype G-T-A-A-T (allele sequence: rs2228611, rs16999593, rs7253062, rs2336691 and rs4804494) and the risk of CAD associated. This study suggests that DNA methylation plays an important role in the pathogenesis of coronary heart disease. DNA, peripheral blood leukocytes may reduce the overall level of methylation is a potential risk factor for coronary heart disease; coronary heart disease is an important step in the pathological process of monocyte foam by methylation regulation of lipid metabolism, SREBP-1 gene promoter methylation abnormalities of the normal expression of the gene polymorphism of DNMT1 gene; the association with coronary heart disease.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R541.4;R440

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