本文選題：右室心尖部起搏 + 三度房室傳導阻滯��； 參考：《北京協(xié)和醫(yī)學院》2017年博士論文

【摘要】：背景三度房室傳導阻滯(Ⅲ atrioventricular block,Ⅲ AVB)是一種常見的緩慢性心律失常,缺乏有效的治療藥物,因此心臟永久起搏器植入是治療Ⅲ AVB的主要方法。目前,多項研究表明右心室心尖部(Right ventricular apical,RVA)起搏可以導致心臟電傳導和機械傳導異常,對心肌細胞代謝和血流灌注造成不利影響,引起心臟重塑,血流動力學紊亂和機械功能減退,最終導致心力衰竭(Heart failure,HF)。此外,心力衰竭的發(fā)生是遺傳學因素和環(huán)境因素共同作用的結果,遺傳因素可以影響某些致病因素導致心力衰竭的易感性、疾病的發(fā)展速度以及對于藥物治療的反應性。目的通過對發(fā)生RVA起搏術后心力衰竭的Ⅲ AVB患者進行臨床隨訪和候選基因遺傳學檢測,采用慢病毒轉染大鼠乳鼠原代心肌細胞(Neonatal rat cardiomyocytes,NRCMs)模型,探索RVA起搏后心力衰竭的遺傳學和致病機制。方法連續(xù)入選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(平均年齡57.4± 18.4歲,465例男性),臨床隨訪發(fā)現(xiàn)10例患者為RVA起搏導致的心力衰竭,同時篩選20例年齡、性別相匹配且未發(fā)生心力衰竭的對照組患者,進行心肌病和心律失常相關候選基因遺傳學檢測。構建Lamin A/C野生型和突變型慢病毒載體,轉染NRCMs,應用PCR、蛋白電泳及細胞免疫熒光檢測特定基因、蛋白的表達與定位;對慢病毒轉染細胞模型進行血清饑餓處理,運用末端標記法(TUNEL)檢測細胞凋亡率;采用Label-free蛋白組學檢測方法分析Lamin A/C野生組與突變組細胞之間的差異蛋白,探究Lamin A/C突變導致RVA起搏后心力衰竭的分子學致病機制。結果臨床隨訪研究發(fā)現(xiàn),877例三度房室傳導阻滯患者中有31例患者因嚴重性難治性心衰再次入院治療,其中10例(平均年齡47.6±10.0歲,4例男性)患者診斷為起搏器術后心力衰竭。候選基因篩查發(fā)現(xiàn)該10例心衰患者均攜帶心肌病致病基因變異,其中3例患者攜帶LMNA基因變異;20例對照組患者中有14例患者未發(fā)現(xiàn)基因變異,6例患者攜帶SCN5A基因突變。采用Lamin A/C慢病毒轉染大鼠乳鼠原代心肌細胞,突變組(LaminA/CR216C和L379F)細胞核大小不一,形狀不規(guī)則,綠色熒光聚集成團,分布不均。此外,電壓門控型鈉離子通道1.5亞型(Navl.5)和縫隙連接蛋白43亞型(Cx43)在野生組和突變組NRCMs中表達和定位無明顯差異。Label-free蛋白組學檢測共發(fā)現(xiàn)144個差異蛋白,其中90個上調蛋白;差異蛋白進行PANTHER分析,核酸結合蛋白為最多的一類蛋白(21%);分子功能分析顯示主要集中在結合(35%)和代謝功能(31%)方面。DAVID、REACTOM、PATHER通路分析顯示差異蛋白主要參與PI3K-Akt和Ras等凋亡相關信號通路。TUNEL法檢測發(fā)現(xiàn)血清饑餓24小時后,與野生組細胞相比,突變組細胞凋亡率顯著升高。結論心肌病致病基因突變是導致患者發(fā)生三度房室傳導阻滯和心力衰竭的遺傳學因素,RVA起搏作為額外刺激可能會加速心力衰竭的發(fā)生發(fā)展。Lamin A/C基因突變(R216C和L379F)可能通過影響Lamin A/C蛋白定位、核酸蛋白結合等途徑參與凋亡信號通路,導致心肌細胞凋亡,最終導致心力衰竭。此外,基因檢測有利于發(fā)現(xiàn)心衰發(fā)生危險性較高的患者,進行臨床預警,闡釋相關基因的致病機制,探索相應治療措施。背景左室致密化不全性心肌病(Left ventricular noncommpaction cardiomyopathy,LVNC)是繼擴張型心肌病、肥厚型心肌病之后的第三種常見的遺傳性心肌病,人群中的發(fā)病率約0.05%—0.3%,以顯著的、與左室心腔相通的心室內肌小梁和深陷的小梁間隱窩或血竇為特征。該畸形可表現(xiàn)出一系列臨床并發(fā)癥,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是一種多基因遺傳病,通常缺乏特定的臨床表型和基因表型相關性。既往研究報道多個基因如TAZ,DNTA,LDB3,YWHAE,MIB1,PRDM16以及肌小節(jié)基因與LVNC有關。通常認為多個致病基因共同參與了 LVNC的病理生理學通路,但是潛在的分子學致病機制并未明了。目的探究漢族LVNC患者的臨床特征、長期隨訪結局和遺傳學檢測結果。方法自2006年3月至2016年3月,連續(xù)入選于阜外醫(yī)院(中國,北京)就診的漢族LVNC病人。入選標準包括超聲心動圖檢查診斷標準和心臟核磁共振成像(Magnetic Resonance Imaging,MRI)檢查診斷標準。詳細地采集每例先證者及其家屬的臨床資料,記錄了患者的病史、超聲心動圖、心臟MRI和心電圖、藥物或者器械治療。對患者進行電話或門診隨訪,隨訪至2016年8月。主要終點事件包括: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進行候選基因遺傳學檢測,候選基因共20個,包括:DTNA,CASQ2,LDB3,GATA4,ACTA1,HCN4,MYH7,LMNA,TNNT2,MYH7B,MIB1,NEXN,PRDM16,NNT,TPM1,RYR2,MYBPC3,YWHAE,TAZ,ACTN2。結果本研究共納入散發(fā)的中國大陸漢族患者222例(149男性,平均年齡43±14歲),其147例患者首次就診時已有心力衰竭,該心衰人群較無心衰患者年齡更高。共41例患者診斷有室性心動過速,16例患者有血栓病史。經(jīng)過34個月的臨床隨訪,32例患者發(fā)生死亡,其中14例患者為心源性猝死,此外還有16例患者進行了心臟移植手術。對總人群進行全因死亡率的多重回歸分析顯示,死亡或心臟移植手術的危險因素有NYHA Ⅲ/Ⅳ(風險比(hazard ratio,HR)3.502;95%置信區(qū)間(confidence interval,CI)1.593-7.694;p0.001),LVEF(HR 0.925;95%CI 0.895-0.957;p0.001)。此外,年齡(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)為SCD的獨立預測因素。對心衰患者進行連續(xù)性超聲心動圖檢查,結果顯示與基線LVEF相比,幸存者隨訪后 LVEF 顯著升高(%,33.7±8.0 vs.37.2±12.2,P0.001),總 LVNC人群的LVEF亦可見相似的結果(%,41.2±12.8 vs.44.5±44.5±13.3,p=0.020)。此外,本研究對32例LVNC患者(24例男性,平均年齡35±17歲)進行了候選基因遺傳學檢測,結果顯示26例(81.3%)患者攜帶LVNC相關基因變異,其中5例患者攜帶兩個或多個基因變異,常見的基因變異為 MYH7B(18.8%),RYR2(15.3%),PRDM16(12.5%)and MYBPC3(12.5%)。由于接受基因檢測的患者數(shù)量較少,本研究并未發(fā)現(xiàn)LVNC患者存在特定的基因型和表型相關性。結論本研究顯示LVNC作為一種多基因遺傳病,具有慢性進展性病程。隨著疾病發(fā)展,LVNC患者可逐漸出現(xiàn)心臟結構和功能惡化,同時可合并室性心律失常、心源性猝死、心衰和血栓栓塞等并發(fā)癥,患者的死亡危險性較高,部分需要進行心臟原位移植挽救生命。NYHA Ⅲ/Ⅳ、低LVEF是LVNC患者出現(xiàn)死亡或心臟原位移植手術的獨立危險因子,此外高齡、基線LVEF較低的心衰患者具有發(fā)生心源性猝死的高度危險性。然而,合理的抗心衰藥物或器械治療可以顯著改善LVNC心衰患者的心臟結構和功能,進而改善臨床預后。今后需要深入研究LVNC患者的遺傳性背景,探究相關基因突變的致病機制,以利于進行臨床預警和治療。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R541;R542.2

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