【作者】 申康均；

【导师】 周新民；

【作者基本信息】 中南大学 ， 外科学， 2012， 博士

【摘要】 风湿性心脏病是一种由于A组溶血性链球菌感染引起的自身免疫性疾病,是引起慢性心衰常见的病因之一。心室泵功能异常,引起血流动力学障碍,心肌收缩力减弱,体循环和肺循环淤血,心脏的血液输出量减少,不能搏出与静脉回流及全身各脏器组织代谢所需相匹配的血液供给,并由此产生一系列临床临床症状和体征。在心衰的发生和进展中,常伴有心肌细胞的肥大性改变和心脏结构性改变,心脏间质纤维化、炎症因子积聚等多种病理变化。风湿性心脏病引起心衰的发病机制较为复杂,涉及多方面的分子病理机制变化。近年来研究发现表观遗传机制参与了心血管疾病的发生和发展过程,表观遗传调控在其中的作用机制逐渐被揭示。表观遗传调控主要包括DNA甲基化和组蛋白修饰,其中DNA甲基化是目前表观遗传学领域研究的热点。研究认为与心血管疾病相关基因的启动子区甲基化状态改变影响了基因的表达水平；此外,基因组DNA整体甲基化水平和DNA甲基化转移酶水平的高低变化也参与了疾病的进程和转归,上述几种异常的甲基化状态被称为“甲基化异常”。目前在冠心病、动脉粥样硬化和先天性心脏病中均观察到这种甲基化异常的状态,而在风湿性心脏病-心衰中表观遗传学相关机制仍处于空白。因此,我们第一部分首先采用实时定量RT-PCR技术检测风湿性心脏病患者和健康对照者右心房心肌组织中与风湿性心脏病-心衰发病密切相关的三个基因BNP、NPRA和ICAM-1mRNA的表达情况,再采用亚硫酸氢钠测序PCR技术检测各基因启动子区各个CG位点的甲基化变化情况,并与临床资料进行分析；第二部分采用ELISA法检测风湿性心脏病患者和健康对照者右心房心肌组织中基因组DNA整体甲基化状况,并探讨其与临床资料之间的关系：第三部分采用实时定量RT-PCR技术检测风湿性心脏病患者和健康对照者右心房心肌组织中DNA甲基化转移酶基因mRNA的表达情况,并与基因组DNA整体甲基化状况和临床资料进行相关分析,从而系统的观察风湿性心脏病-心衰中DNA甲基化异常情况,以期发现风湿性心脏病-心衰过程中的表观遗传学作用机制和特点。第一部分风湿性心脏病-心衰患者心肌组织中脑钠肽基因及其受体和炎症基因的表达及启动子区甲基化水平研究目的观察三种心衰相关基因在风湿性心脏病患者和健康对照者右心房心肌组织中的表达情况,并检测其启动子区DNA甲基化状态,探讨风湿性心脏病-心衰中相关基因表达改变的表观遗传学机制。方法：采用实时定量RT-PCR技术从nRNA水平检测风湿性心脏病患者和健康对照者右心房心肌组织中与心衰密切相关的三个基因BNP、NPRA和ICAM-1的表达情况；采用亚硫酸氢钠测序技术检测风湿性心脏病患者和健康对照组右心房心肌组织中上述三个基因的启动子区甲基化情况,并分析其与临床参数之间的关系。结果：与健康对照组相比,RHD患者右心房心肌组织中BNP基因和ICAM-1基因的mRNA表达水平显著升高(BNP,p<0.001；ICAM-1, p<0.001);而NPRA基因的mRNA表达水平则明显降低(p=0.044)。随着RHD患者病情进展,上述基因的表达水平进一步改变。与NYHAⅡ级组相比,NYHA Ⅲ级组患者右心房心肌组织中BNP基因和ICAM-1基因的mRNA表达水平显著增高(BNP,p<0.001; ICAM-1, p=0.004); NPRA基因的mRNA表达水平显著降低(p=0.001)。与健康对照组相比,RHD患者右心房心肌组织中BNP基因和NPRA基因启动子区的甲基化水平显著增高(BNP,p<0.001；NPRA, p<0.001);而ICAM-1基因启动子区的甲基化水平显著降低(p=0.004)。随着RHD患者病情进展,上述基因启动子区的甲基化水平进一步改变。与NYHAⅡ级组相比,NYHAIII级组患者右心房心肌组织中BNP基因和NPRA基因的启动子区甲基化水平显著增高(BNP,p=0.005:NPRA,p<0.001)；ICAM-1基因启动子区的甲基化水平显著降低(p=0.004)。BNP基因启动子序列甲基化水平与BNP基因的mRNA水平呈显著正相关(r=0.680,P<0.001)；而NPRA基因和ICAM-1基因启动子序列甲基化水平与NPRA基因和ICAM-1的mRNA水平呈显著负相关(NPRA,r=-0.233,P=0.041；ICAM-1,r=-0.459,P<0.001)。三个基因启动子区的甲基化水平与患者年龄均无相关性(P>0.05)。不同性别间、房颤组和无房颤组患者间三个基因启动子区的甲基化水平均无明显差别(P>0.05)。结论：风湿性心脏病患者右心房心肌组织中BNP基因、NPRA基因和ICAM-1基因mRNA的表达发生改变,改变的程度与疾病的严重程度和疾病进展相一致；上述基因启动子区的甲基化水平变化可能是其mRNA表达改变的重要机制,DNA甲基化参与了风湿性心脏病-心衰的发生和发展过程。第二部分风湿性心脏病-心衰患者心肌组织中基因组DNA整体甲基化水平及其临床意义的研究目的：检测风湿性心脏病患者与健康对照者右心房心肌组织中基因组DNA整体甲基化水平,并分析其与临床资料间的关系,探寻基因组DNA整体甲基化状态与风湿性心脏病-心衰之间的关系。方法：应用5-甲基胞嘧啶(5-MC)特异性抗体,采用ELISA法,检测风湿性心脏病患者与健康对照者右心房心肌组织中基因组DNA整体甲基化状态,并分析其与临床资料间的关系。结果：与健康对照组相比,风湿性心脏病患者右心房心肌组织中基因组DNA整体甲基化水平明显增高(p=0.044)；而且随着病情进展、心功能的恶化,基因组DNA整体甲基化水平明显增高,NHYA Ⅲ级组患者右心房心肌组织中基因组DNA整体甲基化水平显著高于NYHA Ⅱ级组患者(p=0.029)。基因组DNA整体甲基化水平与年龄呈显著的正相关性,随着年龄的增长,基因组DNA整体甲基化水平也逐渐增高,(r=0.326,p=0.005),在不同年龄段的对比中(小于40岁、40～50岁,50～60岁和60岁以上),60岁以上年龄组患者右心房心肌组织中的基因组DNA整体甲基化水平明显高于其他三组(p=0.006)。不同性别间、房颤组和无房颤组患者间基因组DNA总体甲基化水平均无明显差别(P>0.05)。结论：风湿性心脏病患者右心房心肌组织中基因组DNA整体呈现高甲基化改变,随着心衰的进展,基因组DNA整体甲基化程度逐渐增高；基因组DNA整体甲基化水平与年龄呈正相关,在高龄组(>60岁)中基因组DNA整体甲基化水平增高更为显著。第三部分风湿性心脏病-心衰患者心肌组织中DNA甲基化转移酶表达及其临床意义的研究目的：观察DNA甲基化转移酶(DNMTs)在风湿性心脏病患者与健康对照者右心房心肌组织中表达情况,并结合DNA甲基化变化情况和临床资料进行分析,探讨DNA甲基化转移酶在风湿性心脏病-心衰异常甲基化过程中的作用。方法：采用实时定量RT-PCR技术从mRNA水平检测风湿性心脏病患者与健康对照者右心房心肌组织中DNMT1、DNMT3a和DNMT3b表达情况,并分析其与基因组DNA整体甲基化状况和临床资料间的关系。结果：与健康对照组相比,风湿性心脏病患者右心房心肌组织中DNMT1、DNMT3a和DNMT3b mRNA表达水平明显增高(DNMT1, p=0.02；DNMT3a,p=0.043；DNMT3b,p=0.035):且随着病情进展,DNMTs的表达水平逐渐升高,与NYHA Ⅱ级组相比,NHYA Ⅲ级组患者右心房心肌组织中DNMT1、DNMT3a和DNMT3b mRNA表达水平明显增高(DNMT1,p=0.019；DNMT3a,p=0.019；DNMT3b, p=0.002)。DNMT1与DNMT3a.DNMT3a与DNMT3b之间的mRNA表达存在显著的正相关性(DNMT1与DNMT3a,r==0.618,p<0.001； DNMT3a与DNMT3b,r=0.509,p<0.001)。Bivariate相关性分析显示在三种DNMTs中,仅DNMT1mRNA的表达水平与基因组整体DNA甲基化水平呈显著正相关(DNMT1,r=0.350,p=0.002； DNMT3a,r=0.221,p=0.053；DNMT3b,r=0.181,p:0.115)；DNMT1、DNMT3a和DNMT3b mRNA表达与年龄、性别和合并房颤均无明显关系。结论：风湿性心脏病-心衰患者右心房心肌组织中三种DNMTs呈高表达状态,且随着心衰的进展,DNMTs mRNA表达水平逐渐升高；三种DNMTs mRNA表达之间存在相关性,其中DNMT1mRNA的表达增高与风湿性心脏病-心衰中基因组DNA整体高甲基化的关系密切。更多还原

【Abstract】 Rheumatic heart disease (RHD) is an autoimmune disease due to group A hemolytic streptococcus infection, which is the common cause of chornic heart failure (CHF). Ventricular pump dysfunction caused by abnormal hemodynamic disorders, decreased myocardial contractility, systemic and pulmonary circulation congestion, the blood output of heart is reduced and cannot pump out the venous retune and the various organs of the body tissue metabolism required to match the blood supply, which will generate a series of signs and symptoms. A variety of pathological changes such as hypertrophic changes of the myocardial cells, cardiac structural changes, cardiac interstitial fibrosis and inflammatory factors accumulation are associated with the development and progression of heart failure. The pathogenesis of heart failure caused by rheumatic heart disease is more complex and involving a wide range of molecular pathological changes. Recent studies have found that epigenetic mechanisms are involved in the onset and development of cardiovascular disease and are gradually being revealed. Epigenetic regulation include DNA methylation and histone modifications, in which DNA methylation is a hotspot of current research in the field of epigenetics. Studies show that gene expression was caused by the changes of cardiovascular disease-related gene promoter methylation status. In addition, the level of global genomic DNA methylation level and DNA methyltransferase is also involved in the process and outcome of disease. The three abnormal methylation status knows as "methylation imbalance". This kind of methylation imbalance is observed in coronary heart disease (CAD), atherosclerosis and congenital heart disease (CHD), however, the related epigenetic mechanism is still remains unknown in rheumatic heart disease. In this study, we firstly detected the mRNA expressions and the promoter methylation levels of three HF-related genes (BNP, NPRA and ICAM-1) in right atrial myocardial tissue of RHD patients and healthy controls by using real-time RT-PCR and the Bisulfite sequencing PCR. Then we measured the global DNA methylation status by ELISA and finally detected the expressions of DNMTs mRNA by real-time RT-PCR. We also explore the relationship between the clinical data and the above parameters meanwhile in order to discover the mechanisms and characteristics of epigenetics in rheumatic heart disease. Part I The inRNA expression levels and promoter methylation patterns of three HF-related genes in myocardium tissue of RHD-HF patientsObjective:To investigate the mRNA expression levels and the promoter methylation patterns of three HF-related genes and potential clinical significance in right atrial myocardium tissue of RHD-HF patients.Methods:Real-time RT-PCR and Bisulfite sequencing PCR (BSP+Sequencing) were performed for evaluated the mRNA expression levels and promoter region methylation patterns of three HF-related genes (BNP, NPRA and ICAM-1) in right atrial myocardial tissue of RHD patients and healthy controls. The correlation between the three HF-related genes mRNA, promoter methylation levels and clinical data were analyzed.Results:Compared with healthy controls, the mRNA expression levels of BNP and ICAM-1gene were significantly increased in right atrial myocardial tissue of RHD patients (BNP, p<0.001; ICAM-1, p <0.001); the mRNA expression level of NPRA gene was significantly reduced (p=0.044). The mRNA expressions of these genes were further changes with the progression of RHD disease. Compared with NYHA Ⅱ group, the mRNA expression levels of BNP and ICAM-1gene were significantly increased in NYHA III group (BNP, p<0.001; ICAM-1, p=0.004); the mRNA expression level of NPRA gene was significantly reduced (p=0.001). For promoter methylation patterns, compared with healthy controls, the promoter methylation levels of BNP and NPRA genes were significantly increased (BNP, p<0.001; NPRA, p<0.001). The promoter methylation level of ICAM-1was significantly reduced (p=0.004). The promoter methylation levels of these genes were further changes with the progression of RHD disease. Compared with NYHA Ⅱ group, the promoter methylation levels of BNP and NPRA gene were significantly increased in NYHA Ⅲ group (BNP, p=0.005; NPRA, p <0.001); the promoter methylation level of NPRA gene was significantly reduced (p=0.004). The promoter methylation level and the mRNA expression level of BNP gene had positive correlation (r=0.680, P <0.001). The promoter methylation level and the mRNA expression level of NPRA gene and ICAM-1gene had negative correlation (NPRA, r=-0.233, P=0.041ICAM-1, r=-0.459, P<0.001). There were no correlations between mRNA expression levels of the three HF-related genes with age, sex and the occurrence of atrial fibrillation (p>0.05).Conclusion:Our findings suggest that the mRNA expression levels of BNP, NPRA and ICAM-1gene were changed in RHD-HF patients and consistent with the severity of the disease and disease progression. The promoter methylation levels could play an important role in these expression level changes, DNA methylation is involved in the development of RHD-HF. Part II Global DNA methylation status and its clinic significance in myocardium tissue of RHD-HF patientsObjective:To evaluate the global DNA methylation status in right atrial myocardial tissue of RHD-HF patients.Methods:ELISA like method with an antibody against5-methylcytosi.ne (5-MC) were performed to observe the global DNA methylation status in right atrial myocardial tissue of RHD patients and healthy controls. The results were compared with the clinical data.Results:Compared with healthy controls, the global DNA methylation status were significantly higher in right atrial myocardial tissue of RHD patients (p=0.044), and the global DNA methylation status were further increased with the progression of RHD disease. Compared with NYHA II group, NYHA III group had higher global DNA methylation status (p=0.029). The global DNA methylation status had positive correlation with age, the global DNA methylation status gradually increased with age increased. Comparison of different ages (<40years,40-50years,50-60years and>60years), the global DNA methylation status of60-year-old age group was significantly higher than the other three age groups. There were no correlations between global DNA methylation status with sex and the occurrence of atrial fibrillation (p>0.05).Conclusion:Global DNA hypermethylation was observed in right atrial myocardial tissue of RHD patients compared with healthy controls. The global DNA methylation status was gradually increased with the progression of disease. The global DNA methylation status had positive correlation with age; the global DNA methylation status of60-year-old age group was significantly higher than the other age groups. Part III The mRNA expressions of DNMTs and clinic significance in myocardium tissue of RHD-HF patientsObjective:To investigate the mRNA expression of DNMTs gene and its potential clinical significance in right atrial myocardial tissue of RHD-HF patients.Methods:The real-time RT-PCR was performed to evaluate the expression of the DNMTs. The correlations between DNMTs mRNA expression with global DNA methylation status and clinical data were analyzed.Results:Compared with healthy controls, the mRNA expression levels of all three DNMTs were significantly higher in right atrial myocardial tissue of RHD patients.(DNMT1, p=0.02; DNMT3a, p=0.043; DNMT3b, p=0.035) and the mRNA expression levels of all three DNMTs were further increased with the progression of RHD disease, Compared with NYHA Ⅱ group, NYHA Ⅲ group had higher mRNA expression levels of the three DNMTs (DNMT1, p=0.019; DNMT3a, p=0.019; DNMT3b, p=0.002). These had positive correlations between mRNA expression levels of DNMT1and DNMT3a, DNMT3a and DNMT3b (DNMT1and DNMT3a, r=0.618, p<0.001; DNMT3a and DNMT3b, r=0.509, p<0.001). Moreover, a significant positive correlation was found between DNMT1mRNA levels and the OD value of global methylation levels (r=0.350, p=0.002). This correlation did not occur between OD values and DNMT3a or DNMT3b mRNA expression levels (r=0.221, p=0.053, for DNMT3a; r=0.181, p=0.115, for DNMT3b). There were no correlations between mRNA expression levels of DNMTs with age, sex and the occurrence of atrial fibrillation (p>0.05).Conclusion:DNMTs genes were over-expressed in right atrial myocardial tissue of RHD patients and the over-expressions of three DNMTs were further increased with the progression of RHD disease. These had correlations between the three DNMTs and DNMT1was closely related with the global DNA hypermethylation status in RHD-HF patients.更多还原