右室心尖部起搏后心力衰竭和左室致密化不全型心肌病的临床特征和致病机制研究

发布时间：2018-06-19 08:49

  本文选题：右室心尖部起搏 + 三度房室传导阻滞　； 参考：《北京协和医学院》2017年博士论文

【摘要】：背景三度房室传导阻滞(Ⅲ atrioventricular block,Ⅲ AVB)是一种常见的缓慢性心律失常,缺乏有效的治疗药物,因此心脏永久起搏器植入是治疗Ⅲ AVB的主要方法。目前,多项研究表明右心室心尖部(Right ventricular apical,RVA)起搏可以导致心脏电传导和机械传导异常,对心肌细胞代谢和血流灌注造成不利影响,引起心脏重塑,血流动力学紊乱和机械功能减退,最终导致心力衰竭(Heart failure,HF)。此外,心力衰竭的发生是遗传学因素和环境因素共同作用的结果,遗传因素可以影响某些致病因素导致心力衰竭的易感性、疾病的发展速度以及对于药物治疗的反应性。目的通过对发生RVA起搏术后心力衰竭的Ⅲ AVB患者进行临床随访和候选基因遗传学检测,采用慢病毒转染大鼠乳鼠原代心肌细胞(Neonatal rat cardiomyocytes,NRCMs)模型,探索RVA起搏后心力衰竭的遗传学和致病机制。方法连续入选1987年1月到2013年12月因ⅢAVB行永久性起搏器植入的患者877例(平均年龄57.4± 18.4岁,465例男性),临床随访发现10例患者为RVA起搏导致的心力衰竭,同时筛选20例年龄、性别相匹配且未发生心力衰竭的对照组患者,进行心肌病和心律失常相关候选基因遗传学检测。构建Lamin A/C野生型和突变型慢病毒载体,转染NRCMs,应用PCR、蛋白电泳及细胞免疫荧光检测特定基因、蛋白的表达与定位;对慢病毒转染细胞模型进行血清饥饿处理,运用末端标记法(TUNEL)检测细胞凋亡率;采用Label-free蛋白组学检测方法分析Lamin A/C野生组与突变组细胞之间的差异蛋白,探究Lamin A/C突变导致RVA起搏后心力衰竭的分子学致病机制。结果临床随访研究发现,877例三度房室传导阻滞患者中有31例患者因严重性难治性心衰再次入院治疗,其中10例(平均年龄47.6±10.0岁,4例男性)患者诊断为起搏器术后心力衰竭。候选基因筛查发现该10例心衰患者均携带心肌病致病基因变异,其中3例患者携带LMNA基因变异;20例对照组患者中有14例患者未发现基因变异,6例患者携带SCN5A基因突变。采用Lamin A/C慢病毒转染大鼠乳鼠原代心肌细胞,突变组(LaminA/CR216C和L379F)细胞核大小不一,形状不规则,绿色荧光聚集成团,分布不均。此外,电压门控型钠离子通道1.5亚型(Navl.5)和缝隙连接蛋白43亚型(Cx43)在野生组和突变组NRCMs中表达和定位无明显差异。Label-free蛋白组学检测共发现144个差异蛋白,其中90个上调蛋白;差异蛋白进行PANTHER分析,核酸结合蛋白为最多的一类蛋白(21%);分子功能分析显示主要集中在结合(35%)和代谢功能(31%)方面。DAVID、REACTOM、PATHER通路分析显示差异蛋白主要参与PI3K-Akt和Ras等凋亡相关信号通路。TUNEL法检测发现血清饥饿24小时后,与野生组细胞相比,突变组细胞凋亡率显著升高。结论心肌病致病基因突变是导致患者发生三度房室传导阻滞和心力衰竭的遗传学因素,RVA起搏作为额外刺激可能会加速心力衰竭的发生发展。Lamin A/C基因突变(R216C和L379F)可能通过影响Lamin A/C蛋白定位、核酸蛋白结合等途径参与凋亡信号通路,导致心肌细胞凋亡,最终导致心力衰竭。此外,基因检测有利于发现心衰发生危险性较高的患者,进行临床预警,阐释相关基因的致病机制,探索相应治疗措施。背景左室致密化不全性心肌病(Left ventricular noncommpaction cardiomyopathy,LVNC)是继扩张型心肌病、肥厚型心肌病之后的第三种常见的遗传性心肌病,人群中的发病率约0.05%—0.3%,以显著的、与左室心腔相通的心室内肌小梁和深陷的小梁间隐窝或血窦为特征。该畸形可表现出一系列临床并发症,如心力衰竭、心律失常包括心源性猝死(sudden cardiac death,SCD)和血栓栓塞。此外,LVNC是一种多基因遗传病,通常缺乏特定的临床表型和基因表型相关性。既往研究报道多个基因如TAZ,DNTA,LDB3,YWHAE,MIB1,PRDM16以及肌小节基因与LVNC有关。通常认为多个致病基因共同参与了 LVNC的病理生理学通路,但是潜在的分子学致病机制并未明了。目的探究汉族LVNC患者的临床特征、长期随访结局和遗传学检测结果。方法自2006年3月至2016年3月,连续入选于阜外医院(中国,北京)就诊的汉族LVNC病人。入选标准包括超声心动图检查诊断标准和心脏核磁共振成像(Magnetic Resonance Imaging,MRI)检查诊断标准。详细地采集每例先证者及其家属的临床资料,记录了患者的病史、超声心动图、心脏MRI和心电图、药物或者器械治疗。对患者进行电话或门诊随访,随访至2016年8月。主要终点事件包括:全因死亡、SCD和非心源性猝死(Non-sudden cardiac death,NSCD)。采集每例患者的外周血进行候选基因遗传学检测,候选基因共20个,包括:DTNA,CASQ2,LDB3,GATA4,ACTA1,HCN4,MYH7,LMNA,TNNT2,MYH7B,MIB1,NEXN,PRDM16,NNT,TPM1,RYR2,MYBPC3,YWHAE,TAZ,ACTN2。结果本研究共纳入散发的中国大陆汉族患者222例(149男性,平均年龄43±14岁),其147例患者首次就诊时已有心力衰竭,该心衰人群较无心衰患者年龄更高。共41例患者诊断有室性心动过速,16例患者有血栓病史。经过34个月的临床随访,32例患者发生死亡,其中14例患者为心源性猝死,此外还有16例患者进行了心脏移植手术。对总人群进行全因死亡率的多重回归分析显示,死亡或心脏移植手术的危险因素有NYHA Ⅲ/Ⅳ(风险比(hazard ratio,HR)3.502;95%置信区间(confidence interval,CI)1.593-7.694;p0.001),LVEF(HR 0.925;95%CI 0.895-0.957;p0.001)。此外,年龄(HR 1.073,95%CI 1.018-1.130;p=0.009)和 LVEF(HR 0.881;95%CI 0.809-0.958;p=0.003)为SCD的独立预测因素。对心衰患者进行连续性超声心动图检查,结果显示与基线LVEF相比,幸存者随访后 LVEF 显著升高(%,33.7±8.0 vs.37.2±12.2,P0.001),总 LVNC人群的LVEF亦可见相似的结果(%,41.2±12.8 vs.44.5±44.5±13.3,p=0.020)。此外,本研究对32例LVNC患者(24例男性,平均年龄35±17岁)进行了候选基因遗传学检测,结果显示26例(81.3%)患者携带LVNC相关基因变异,其中5例患者携带两个或多个基因变异,常见的基因变异为 MYH7B(18.8%),RYR2(15.3%),PRDM16(12.5%)and MYBPC3(12.5%)。由于接受基因检测的患者数量较少,本研究并未发现LVNC患者存在特定的基因型和表型相关性。结论本研究显示LVNC作为一种多基因遗传病,具有慢性进展性病程。随着疾病发展,LVNC患者可逐渐出现心脏结构和功能恶化,同时可合并室性心律失常、心源性猝死、心衰和血栓栓塞等并发症,患者的死亡危险性较高,部分需要进行心脏原位移植挽救生命。NYHA Ⅲ/Ⅳ、低LVEF是LVNC患者出现死亡或心脏原位移植手术的独立危险因子,此外高龄、基线LVEF较低的心衰患者具有发生心源性猝死的高度危险性。然而,合理的抗心衰药物或器械治疗可以显著改善LVNC心衰患者的心脏结构和功能,进而改善临床预后。今后需要深入研究LVNC患者的遗传性背景,探究相关基因突变的致病机制,以利于进行临床预警和治疗。
[Abstract]:Background three degree atrioventricular block (III atrioventricular block, III AVB) is a common slow arrhythmia and lacks effective therapeutic drugs. Therefore, permanent cardiac pacemaker implantation is the main method for the treatment of III AVB. At present, a number of studies have shown that the Right ventricular apical (RVA) pacing can lead to cardiac electricity. The abnormalities of conduction and mechanical conduction have adverse effects on the metabolism and blood perfusion of cardiac myocytes, causing cardiac remodeling, hemodynamic disorders and mechanical dysfunction, and ultimately leading to heart failure (Heart failure, HF). In addition, the occurrence of heart failure is the result of the common effect of genetic and environmental factors, and genetic factors can affect a certain extent. Some pathogenic factors lead to the susceptibility to heart failure, the speed of development of the disease and the response to drug treatment. Objective to follow up the clinical follow-up and candidate gene genetic detection of the patients with heart failure after RVA pacing, and to transfect Neonatal rat cardiomyocytes, NR with lentivirus transfection in rats. CMs) model, explore the genetics and pathogenesis of heart failure after RVA pacing. Methods 877 patients with permanent pacemaker implantation from January 1987 to December 2013 were enrolled in 877 patients (average age 57.4 + 18.4 years, 465 men). Clinical follow-up found that 10 patients were heart failure caused by RVA pacing, and 20 cases of age and sex were screened at the same time. Lamin A/C wild type and mutant lentivirus vector, transfected NRCMs, PCR, protein electrophoresis and cell immunofluorescence were used to detect specific genes, the expression and localization of egg white, and the model of lentivirus transfected cell model. The cell apoptosis rate was detected by the end labeling method (TUNEL), and the differential protein between the Lamin A/C wild group and the mutant group cells was analyzed by the Label-free proteomics method, and the mechanism of the division of the Lamin A/C mutation to the heart failure after RVA pacing was investigated. The results of clinical follow-up study found that 877 cases were three degrees. Of the patients with atrioventricular block, 31 patients were admitted to hospital for severe refractory heart failure, of which 10 cases (average age 47.6 10 years old and 4 male) were diagnosed with heart failure after pacemaker operation. The candidate gene screening found that all the 10 patients with heart failure carry the genetic variation of cardiomyopathy, of which 3 patients carried the LMNA gene mutation. In 20 cases, 14 patients had no genetic variation, and 6 patients carried SCN5A gene mutation. Lamin A/C lentivirus was used to transfect primary cardiomyocytes of rat milk rats. The mutant group (LaminA/CR216C and L379F) had different nuclei size, irregular shape, green fluorescence aggregation and uneven distribution. In addition, voltage gated sodium channels were distributed. The expression and localization of 1.5 subtype (Navl.5) and gap connexin 43 subtype (Cx43) in the wild group and the mutant group NRCMs were not significantly different. 144 differential proteins were found in the.Label-free protein group detection, of which 90 were up to be up protein; the difference protein was analyzed by PANTHER, and the nucleic acid binding protein was the most kind of protein (21%); molecular functional analysis showed that In combination with (35%) and metabolic function (31%),.DAVID, REACTOM, and PATHER pathway analysis showed that the difference protein was mainly involved in PI3K-Akt and Ras apoptosis related signaling pathway.TUNEL detection to detect serum hunger 24 hours later, compared with the wild group cells, the apoptosis rate of the mutant group was significantly increased. Conclusion the mutation of the cardiomyopathy gene is a conclusion. The genetic factors that cause three degrees of atrioventricular block and heart failure in patients, RVA pacing as an additional stimulus may accelerate the development of the.Lamin A/C gene mutation (R216C and L379F), which may be involved in the apoptosis signaling pathway by influencing the localization of Lamin A/C protein, the binding of nucleic acid protein and the pathway of nucleic acid protein binding, leading to cardiomyocyte apoptosis. In addition, gene detection is beneficial to the detection of patients with high risk of heart failure, clinical early warning, interpretation of the pathogenesis of related genes, and the exploration of corresponding treatment measures. Background left ventricular compact and incomplete cardiomyopathy (Left ventricular noncommpaction cardiomyopathy, LVNC) is followed by dilated cardiomyopathy and hypertrophy. The incidence of third common hereditary cardiomyopathy after type cardiomyopathy is about 0.05% - 0.3% in the population. It is marked by the intraventricular trabecula of the left ventricular cavity and the deep trabecular intertrabecular recess or sinus. The malformation can show a series of clinical complications, such as heart failure, and arrhythmia including sudden cardiac death (sudden Cardiac death, SCD) and thromboembolism. In addition, LVNC is a polygenic disease that usually lacks specific clinical phenotypes and genetic phenotypes. Previous studies have reported that multiple genes, such as TAZ, DNTA, LDB3, YWHAE, MIB1, PRDM16, and myocutaneous genes, are associated with LVNC. Generally, multiple genes are considered to be involved in the pathophysiology of LVNC. To explore the clinical characteristics, long-term follow-up outcome and genetic test results of Han LVNC patients. Methods from March 2006 to March 2016, the Han LVNC disease patients were enrolled in Fuwai Hospital (China, Beijing). The criteria included echocardiographic diagnostic criteria. The diagnostic criteria of Magnetic Resonance Imaging (MRI) were examined in detail. The clinical data of each case precursor and his family were collected in detail, the patient's history, echocardiography, cardiac MRI and electrocardiogram, medicine or apparatus were treated. The patients were followed up by telephone or outpatient, followed up to the end of August 2016. SCD and non cardiogenic sudden death (Non-sudden cardiac death, NSCD). The candidate gene genetic testing of the peripheral blood of each patient was collected, including 20 candidate genes, including DTNA, CASQ2, LDB3, GATA4, ACTA1, HCN4. 222 cases (149 men, 43 + 14 years old) were included in the Chinese mainland. 147 patients had heart failure in the first visit. The heart failure population was older than those with no heart failure. 41 patients diagnosed with ventricular tachycardia and 16 patients had a history of thrombus. After 34 months of clinical follow-up, 32 patients died, 14 patients were sudden cardiac death, and 16 patients underwent cardiac transplantation. Multiple regression analysis of total mortality for total population showed that the risk factors for death or heart transplantation were NYHA III / IV (hazard ratio, HR) 3.502; 95% confidence interval (confidence interval, CI) 1.593-7.694; p0.001), LV. EF (HR 0.925; 95%CI 0.895-0.957; p0.001). In addition, independent predictors of age (HR 1.073,95%CI 1.018-1.130; p=0.009) and LVEF (HR 0.881; 95%CI). Continuous echocardiography for patients with heart failure showed that the survivors were significantly increased after follow-up (%, 33.7 + 8). The LVEF of the total LVNC population was also similar (%, 41.2 + 12.8 vs.44.5 + 44.5 + 13.3, p=0.020). In addition, the genetic test of candidate genes was carried out in 32 cases of LVNC patients (24 men, average age 35 + 17 years). The results showed that 26 (81.3%) patients carried LVNC related gene mutations, among which 5 patients carried two or 12.8. The common genetic variation is MYH7B (18.8%), RYR2 (15.3%), PRDM16 (12.5%) and MYBPC3 (12.5%). Because the number of patients receiving gene detection is less, this study does not find that there is a specific genotype and phenotypic correlation in LVNC patients. Conclusion this study shows that LVNC is a polygenic disease with chronic progresses. Course of the disease. With the development of the disease, LVNC patients can gradually deteriorate heart structure and function, and can combine ventricular arrhythmia, sudden cardiac death, heart failure and thromboembolism. The risk of death is higher in patients. Partial heart transplantation is needed to save life.NYHA III / IV, and low LVEF is the death or heart of LVNC patients. Independent risk factors of orthotopic transplantation, in addition to elderly patients with lower baseline LVEF, have a high risk of sudden cardiac death. However, rational anti heart failure or apparatus treatment can significantly improve the cardiac structure and function of patients with LVNC heart failure and further improve the clinical prognosis. Further study of LVNC patients is needed in the future. Genetic background, explore the pathogenesis of gene mutation, so as to facilitate clinical early warning and treatment.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R541;R542.2

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7 柳胜华;左室心肌致密化不全型心肌病分子遗传机制研究[D];北京协和医学院;2013年

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9 黄为;结蛋白相关心肌病发病机制的研究[D];南京医科大学;2003年

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9 曾施;速度矢量成像定量评价心肌病左心长轴功能的研究[D];中南大学;2007年

10 庞洁;Takotsubo心肌病诊治2例[D];浙江大学;2011年

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