外周血白细胞基因组DNA甲基化在心力衰竭中的作用及临床意义

发布时间：2017-12-28 17:31

本文关键词：外周血白细胞基因组DNA甲基化在心力衰竭中的作用及临床意义　出处：《吉林大学》2017年博士论文　论文类型：学位论文

更多相关文章： 心力衰竭 DNA甲基化 白细胞 基因组

【摘要】：心力衰竭(Heart Failure,HF)主要表现在体循环静脉以及肺静脉的淤血。心衰是一个进展性、不可逆性的病理过程。近年来已明确在心力衰竭发生、发展过程中基因表达调控的变化起着重要的作用。DNA甲基化在哺乳动物中是最常见的表观遗传调节机制,有可能引发心衰的发生和发展,但大部分的研究都是取材心肌组织,本研究首次对心衰患者外周血白细胞的基因组DNA甲基化进行分析,明确心衰过程中某些基因的甲基化表达水平。本实验应用简化代表性重亚硫酸盐测序(RRBS)对DNA序列样本进行测序;应用定量MassARRAY甲基化分析来证实心力衰竭组基因序列的甲基化水平和正常对照组之间的不同,随后应用两组样本,我们进行了实时定量PCR检测,RT-qPCR能够进一步明确两组人群不同的基因表达,从而进一步证实RRBS和定量MassARRAY甲基化分析的结果。最后经过酶联免疫吸附测定(ELISA)方法对各个基因相应蛋白的表达水平进行检测。本研究得出以下结果:1.经过RRBS分析,心力衰竭组外周血白细胞共测出了11228个CG重复序列;而在正常对照组外周血白细胞中,共确定其中存在10680个CG重复序列。在两组的CpG重复序列中,本研究共分析出了732个差异性甲基化区域。2.通过定量MassArray甲基化分析的结果显示,对于MPV17L基因,心力衰竭组和正常对照组的三个样本的甲基化水平分别为5.7%,6.7%,6.5%和8.2%,7.8%,8.8%;对于SLC2A1基因,心力衰竭组和正常对照组的三个样本的甲基化水平分别为21.4%,37.9%,14.3%和37.4%,41.9%,34.7%;对于PLEC基因,心力衰竭组和正常对照组的三个样本的甲基化水平分别为83.3%,63.8%,62%和57%,59.1%,60.1%。3.RT-qPCR的结果显示,外周血白细胞三种目的基因中的SLC2A1基因在心力衰竭组患者的表达水平(0.499±0.388)较正常对照组明显升高(0.062±0.041,p0.001);MPV17L基因的表达水平(0.003±0.002)较正常对照组明显升高(0.001±0.001,p=0.003);心力衰竭组患者外周血白细胞PLEC基因的表达水平(0.023±0.014)较正常对照组明显减低(0.064±0.039,p=0.002)。4.SLC2A1基因启动子序列甲基化水平与mRNA表达水平呈显著负相关(r=-0.089,p=0.042);MPV17L基因的mRNA表达水平与MPV17L基因启动子序列甲基化水平呈显著负相关(r=-0.886,p=0.019);PLEC基因序列甲基化水平与该基因的mRNA表达水平呈显著负相关(r=-0.829,p=0.042)。5.心力衰竭患者SLC2A1基因的相关蛋白的表达水平(5286.3±2558.2pg/ml),较正常对照组明显升高(2478.9±614.6 pg/ml,p=0.007);心力衰竭患者白细胞中MPV17L基因的相关蛋白的表达水平为376.1±287.8 pg/ml,较正常对照组明显升高(155.3±64.9 pg/ml,p=0.04);心力衰竭患者白细胞中PLEC基因的相关蛋白的表达水平为(35970.4±17646.3 pg/ml),较正常对照组明显降低(86773.4±47142.4 pg/ml,p=0.03)。结论:1、和既往取材心肌组织的方法相比,本实验研究心衰组和对照组之间外周血白细胞中DNA序列甲基化的不同的方法是非常简单易行的。2、心衰组外周血白细胞中DNA序列的甲基化水平低于正常对照组,有可能是心力衰竭发生和发展的机制之一。3、明确了SLC2A1基因、MPV17L基因和PLEC基因在心衰患者中的表达水平以及相应蛋白的表达水平,为DNA序列甲基化对于心力衰竭发生和发展的作用提供了详细的临床依据。
[Abstract]:Heart failure (Heart Failure, HF) is mainly manifested in the vein of the body and the blood stasis of the pulmonary vein. Heart failure is a progressive and irreversible pathological process. In recent years, the change of gene expression and regulation plays an important role in the process of heart failure and development. DNA methylation in mammals is the most common epigenetic regulatory mechanisms, may lead to the occurrence and development of heart failure, but most of them are based on the analysis of myocardial tissue, DNA methylation for the first time in patients with heart failure and peripheral white blood cells, the level of methylation specific expression of certain genes in chronic heart failure the. The application of simplified representative bisulfite sequencing (RRBS) sequence of DNA sequence samples; using quantitative MassARRAY methylation analysis between different methylation levels to confirmed gene sequence of the failure group and normal control group, followed by the two group samples, we performed quantitative real-time PCR detection, to further clarify the expression of RT-qPCR two groups of different genes, which further confirmed the quantitative analysis of RRBS and MassARRAY methylation results. Finally, the expression level of the corresponding proteins was detected by enzyme linked immunosorbent assay (ELISA). The following results were obtained in this study: 1.. After RRBS analysis, 11228 CG repeats were detected in peripheral blood leukocytes from heart failure group, while 10680 CG repeats were identified in peripheral blood leukocytes of normal control group. In the two groups of CpG repeat sequences, 732 differential methylation regions were analyzed in this study. 2. by quantitative MassArray methylation analysis showed that the MPV17L gene methylation level in a sample of three heart failure group and normal control group were 5.7%, 6.7%, 6.5% and 8.2%, 7.8%, 8.8%; for SLC2A1 gene, methylation level of three samples of heart failure group and normal control group were 21.4% 37.9%, 14.3%, and 37.4%, 41.9%, 34.7%; for PLEC gene, methylation level of three samples of heart failure group and normal control group were 83.3%, 63.8%, 62% and 57%, 59.1%, 60.1%. The results of 3.RT-qPCR showed that peripheral white blood cells of three target genes in SLC2A1 gene expression level in heart failure patients (0.499 + 0.388) was significantly higher than the control (0.062 + 0.041, p0.001); the expression level of MPV17L gene (0.003 + 0.002) compared with the normal control group was significantly higher (0.001. 0.001, p=0.003); the expression level of PLEC in white blood cell in peripheral blood of patients with heart failure were foreign genes (0.023 + 0.014) compared with the normal control group was significantly lower (0.064 + 0.039, p=0.002). 4.SLC2A1 gene promoter methylation level and the expression level of mRNA was negatively correlated (r=-0.089, p=0.042); MPV17L gene expression level of mRNA and MPV17L gene promoter methylation level was negatively correlated (r=-0.886, p=0.019); PLEC gene methylation and the gene expression level of mRNA was negatively correlated (r=-0.829, p=0.042). The expression level of SLC2A1 gene in patients with heart failure related protein 5. of the (5286.3 + 2558.2pg/ml), was obviously higher than normal control group (2478.9 + 614.6 pg/ml, p=0.007); the expression level of MPV17L protein gene of white blood cells in patients with heart failure was 376.1 + 287.8 pg/ml, significantly higher than that in control group (155.3 + 64.9 pg/ml, p=0.04 the expression level of PLEC protein); white blood cells in patients with heart failure gene is (35970.4 + 17646.3 pg/ml), was lower than the control group (86773.4 + 47142.4 pg/ml, p=0.03). Conclusion: 1. Compared with previous methods of myocardial tissue extraction, the methylation of DNA sequence in peripheral blood leukocytes between heart failure group and control group is very simple and feasible. 2. The methylation level of DNA sequence in peripheral blood leukocytes in the heart failure group is lower than that of the normal control group, which may be one of the mechanisms of the occurrence and development of heart failure. 3, we defined the expression level of SLC2A1 gene, MPV17L gene and PLEC gene in heart failure patients and the expression level of corresponding proteins, which provided a detailed clinical basis for the role of DNA sequence methylation in the occurrence and development of heart failure.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R541.6

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