过表达ACE2对犬心房快速起搏所致的心房重构的影响及机制研究

发布时间：2018-04-23 06:58

本文选题：心房颤动 + 心房纤维化　；参考：《重庆医科大学》2016年博士论文

【摘要】：背景:心房颤动(Atrial fibrillation,AF,简称房颤),是临床上最常见且难以治疗、易复发的一种心律失常,已成为人类心血管疾病中的一大流行病。多种疾病例如缺血性、瓣膜性、炎症性和老年退化性心脏疾病、高血压等均易引发房颤,然而目前房颤的治疗效果尚不尽如人意。大量研究已证实肾素-血管紧张素系统(Renin angiotensin system,RAS)在房颤的发生与维持中被不同程度地激活。血管紧张素II(Angiotensin II,Ang II)是RAS最重要的效应分子,具有收缩血管、促进纤维化增生和心肌肥大等效应,它能被血管紧张素转换酶2(Angiotensin converting enzyme 2,ACE2)转化成为有保护性效应的血管紧张素-(1-7)[Angiotensin-(1-7),Ang-(1-7)]。新近研究表明,过表达ACE2能拮抗Ang II介导的心房重构效应。心房重构是房颤发生和维持的基本机制,改良心房基质成为防治房颤的基础环节。有研究表明,Rac1在心肌细胞中可能经上调结缔组织生长因子(Connective Tissue Growth Factor,CTGF)、N-Cadherin及Cx43这一途径促进了心脏纤维化及心房重构;Rad(Ras Associated with Diabetes)GTPase在心脏中能抑制CTGF的转录,经Rho/Rhokinase途径调节细胞骨架的重塑。然而,在房颤中Rac1和Rad GTPase的动态平衡的调节机制尚不明确,过表达ACE2改善心房纤维化的机制需进一步探讨。目的:本研究拟建立犬心房快速起搏模型,应用心房外膜涂抹携带有ACE2基因的腺病毒的方法探讨心房过表达ACE2对改善犬心房快速起搏所致的心房重构的影响及其分子机制。方法:入选28只成年健康的杂种犬(22±2Kg,雌雄不拘),随机分为4组,每组7只:Sham组(假手术组)、AF-Control组(心房快速起搏对照组)、AF-EGFP组(心房快速起搏+涂染Ad-EGFP腺病毒)及AF-ACE2组(心房快速起搏+涂染Ad-ACE2腺病毒)。其中AF-ACE2组与AF-EGFP组犬心房外膜分别涂染带有ACE2和EGFP的腺病毒,Sham组与AF-Control组犬不涂染腺病毒。除Sham组外,AF-Control组、AF-EGFP组和AF-ACE2组均予以持续心房快速起搏(450次/min)。2周后,4组犬行第一次开胸手术及心内电生理检查(Electrophysiological Study,EP),进行基因涂染。转染3周后行第二次开胸手术及EP检查,随后处死,取心房组织标本进行一下研究:应用苏木精-伊红(Hematoxylin-Eosin,HE)染色和天狼猩红染色评价心房快速起搏所致的组织病理学改变;应用免疫印迹技术评价RAS成员AT1R、AT2R、Apelin和Aplnr的表达,以探讨过表达ACE2对RAS系统的影响;应用免疫组织化学方法和免疫印迹技术评价Rac1、Rad GTPase和Gem的表达,应用免疫印迹技术和免疫组织化学荧光方法评价CTGF、α-SMA和N-Cadherin的表达,应用实时荧光定量RT-PCR方法评价c-jun、c-myc和c-fos的基因表达,以探讨过表达ACE2对心房快速起搏所致的心房纤维化重构的影响。结果:两次开胸手术中心内电生理检查结果相比,第二次手术中AF-Control组、AF-EGFP组及AF-ACE2组犬心房ERP均显著降低,AERP频率适应性也显著下降,而Sham组犬心房ERP在基因转染前后无明显变化。同时,AF-EGFP组(4/7 vs.7/7)和AF-Control组(2/7vs.7/7)犬房性心律失常的诱发率明显上升,且持续时间显著延长,而AF-ACE2组犬房性心律失常的诱发率(4/7 vs.3/7)及持续时间均无明显变化,Sham组犬仅能诱发出短暂的房性心律失常。组织学和分子生物学研究结果:HE染色可见实验犬均无明显出血及炎症渗出等。假手术组细胞间隙适中,纤维排列整齐;AF-Control组和AF-EGFP组心房肌纤维排列紊乱,出现明显的挛缩和断裂;而上述异常在AF-ACE2组明显减轻,与AF-Control组和AF-EGFP组相比,转染ACE2后心房肌纤维分布相对整齐,排列大致有序。天狼猩红染色显示AF-Control组和AF-EGFP组心肌间质纤维化明显,纤维组织增生显著,胶原纤维分布广泛,纤维化程度明显强于Sham组和AF-ACE2组。定量分析结果表明AF-ACE2组的胶原面积明显低于AF-Control组和AF-EGFP组(P0.0001),与Sham组无明显差异(P=0.234)。免疫印迹技术评价RAS成员的表达,结果显示AF-Control组和AF-EGFP组AT1R蛋白的表达水平显著高于Sham组和AF-ACE2组,而AT2R蛋白的表达水平在AF-Control组和AF-EGFP组显著降低。Apelin和Aplnr的蛋白表达在AF-Control组和AF-EGFP组显著降低,而过表达ACE2则显著升高了两者的表达水平(P0.05)。免疫印迹技术评价Rac1、Rad GTPase和Gem的表达结果显示:AF-Control组和AF-EGFP组Rac1蛋白的表达水平显著高于Sham组和AF-ACE2组(P0.05)。而Rad GTPase的表达在AF-ACE2组显著升高(P0.0001),与Rad同属于RKG家族的Gem在AF-Control组和AF-EGFP组的表达也显著降低(P0.0001)。免疫组化方法评价Rac1和Gem所得结果与上述免疫印迹法的结果一致。免疫印迹技术评价CTGF、α-SMA和N-Cadherin的表达,结果显示三者在AF-Control组和AF-EGFP组的表达均显著升高,而过表达ACE2则逆转了这一趋势。同时采用免疫组织化学荧光方法检测α-SMA的表达,结果表明α-SMA主要分布在细胞胞浆之中,在AF-Control组和AF-EGFP组中可见大量的绿色荧光细胞且α-SMA的表达强于Sham组和AF-ACE2组,与蛋白印迹的结果相一致。同时,Realtime RT-PCR结果显示c-jun、c-myc和c-fos的基因表达在AF-Control组和AF-EGFP组显著升高,而在AF-ACE2组中过表达ACE2能降低三者的m RNA表达水平。结论:本研究结果表明:(1)过表达ACE2可以降低房颤的诱发率和缩短房颤的持续时间;(2)过表达ACE2调节心房快速起搏所致的RAS失衡,显著下调AT1R的表达,上调AT2R、Apelin和Aplnr的表达;(3)过表达ACE2能调节Rac1和Rad GTPase的动态平衡,显著下调Rac1的表达和上调Rad GTPase、Gem的表达,抑制纤维化相关因子CTGF、α-SMA和N-Cadherin的表达以及下调c-fos、c-jun和c-myc的基因表达,以改善心房纤维化重构。综上所述,本研究结果表明心房过表达ACE2能够通过调节心房快速起搏所致的失衡RAS轴向保护性方向移动、上调Rad GTPase的表达同时下调Rac1的表达、抑制纤维化相关因子的表达这些机制来实现改善心房重构和改良心房基质的目的,从而达到有效降低房颤的诱发率和缩短房颤的持续时间的效果,房颤的防治得到最终获益。
[Abstract]:Background: Atrial fibrillation (AF) is the most common and difficult to treat and relapse arrhythmia in clinic. It has become an epidemic in human cardiovascular disease. Many diseases such as ischemic, valvular, inflammatory and senile degenerative heart disease, hypertension and so on are easy to cause atrial fibrillation. However, it is present The therapeutic effect of atrial fibrillation is not satisfactory. A large number of studies have proved that the Renin angiotensin system (RAS) is activated to varying degrees in the occurrence and maintenance of atrial fibrillation. Angiotensin II (Angiotensin II, Ang II) is the most important effector of RAS, which has contractile vessels, promotes fibrosis and myocardial fertilizer. Big effect, which can be transformed into angiotensin converting enzyme 2 (Angiotensin converting enzyme 2, ACE2) into protective effects of angiotensin - (1-7) [Angiotensin- (1-7), Ang- (1-7)). Recent studies have shown that overexpression of ACE2 can antagonize the atrial remodeling effect mediated by Ang II. Atrial remodeling is the basic mechanism for atrial fibrillation and maintenance. The matrix of the conscience room is the basic link in the prevention and treatment of atrial fibrillation. Some studies have shown that Rac1 may increase the connective tissue growth factor (Connective Tissue Growth Factor, CTGF) in cardiac myocytes, N-Cadherin and Cx43 promote cardiac fibrosis and atrial remodeling; Rad (Ras Associated with) can inhibit the heart in the heart. The transcription of the cytoskeleton is regulated by the Rho/Rhokinase pathway. However, the regulatory mechanism of the dynamic balance of Rac1 and Rad GTPase in atrial fibrillation is not clear. The mechanism for overexpressing ACE2 to improve atrial fibrosis needs to be further explored. Objective: This study is to establish a canine atrial rapid pacing model and use the outer membrane of the atrial membrane to carry a ACE2 gene. The effects and molecular mechanisms of atrial overexpression of ACE2 on atrial remodeling in canine atrial rapid pacing were investigated by adenovirus method. Methods: 28 healthy adult hybrids (22 2Kg, male and female) were randomly divided into 4 groups, group Sham (sham operation group), group AF-Control (atrial rapid pacing control group), AF-EGFP group (heart) Atrial rapid pacing + smearing of Ad-EGFP adenovirus and AF-ACE2 group (atrial rapid pacing + smearing Ad-ACE2 adenovirus). In group AF-ACE2 and AF-EGFP group, the atrial outer membrane was coated with ACE2 and EGFP adenovirus respectively, and Sham group and AF-Control group were not stained with adenovirus. Except Sham group, AF-Control group, AF-EGFP group and group were all sustained atrium. After the rapid pacing (450 /min).2 weeks, the 4 dogs were performed the first thoracotomy and Electrophysiological Study (EP), and the gene smear was performed. After 3 weeks of transfection, second thoracotomy and EP examinations were performed, and then the specimens were executed, and the specimens of the atrium tissue were studied for the use of hematoxylin eosin (Hematoxylin-Eosin, HE) staining and the day. Wolf scarlet staining was used to evaluate the histopathological changes caused by rapid atrial pacing; the expression of AT1R, AT2R, Apelin and Aplnr in RAS members was evaluated by Western blot to explore the effect of ACE2 on RAS system; the expression of Rac1, Rad GTPase and Gem were evaluated by immunohistochemistry and immunoblotting techniques, and Western blot technique was applied. To evaluate the expression of CTGF, alpha -SMA and N-Cadherin, and to evaluate the expression of c-jun, c-myc and c-fos by real time fluorescence quantitative RT-PCR method, the effect of ACE2 on atrial fibrosis remodeling caused by rapid atrial pacing was investigated. Results: the results were compared with the results of electrophysiological examination in the two thoracotomy centers. In group AF-Control, group AF-EGFP and group AF-ACE2, the atrial ERP of the dogs decreased significantly and the frequency of AERP decreased significantly, while the ERP of the dog atrium in the Sham group had no significant changes before and after gene transfection. At the same time, the induced rate of atrial arrhythmia in AF-EGFP group (4/7 vs.7/7) and AF-Control group (2/7vs.7/7) was significantly increased, and the duration was significant. There was no obvious change in the induction rate (4/7 vs.3/7) and duration of atrial arrhythmia in group AF-ACE2. The dogs in group Sham could only induce transient atrial arrhythmias. Histology and molecular biology research results: HE staining showed that there was no obvious bleeding and inflammatory infiltration in the experimental dogs. The sham operation group had moderate intercellular space and fiber arrangement. The atrial fibers in group AF-Control and group AF-EGFP were arranged in disorder and showed obvious contracture and fracture, and the abnormality of the above abnormality was obviously reduced in group AF-ACE2. Compared with group AF-Control and AF-EGFP, the distribution of the fibers in the atrial muscle was relatively neatly and orderly after the transfection of ACE2. Sirius scarlet staining showed the interstitial fibrosis of the AF-Control group and the AF-EGFP group. The proliferation of the fibrous tissue was obvious, the collagen fiber was widely distributed, the fibrosis degree was stronger than the Sham group and the AF-ACE2 group. The quantitative analysis showed that the area of collagen in AF-ACE2 group was obviously lower than that of group AF-Control and AF-EGFP group (P0.0001), and there was no significant difference between group AF-ACE2 and Sham group (P=0.234). The expression of RAS members was evaluated by immunoblotting technique, and the results showed A. The expression level of AT1R protein in group F-Control and AF-EGFP group was significantly higher than that in group Sham and AF-ACE2, but the expression level of AT2R protein in AF-Control group and AF-EGFP group decreased significantly in AF-Control group and AF-EGFP group. The expression of Rac1, Rad GTPase and Gem showed that the expression level of Rac1 protein in AF-Control group and AF-EGFP group was significantly higher than that of Sham and AF-ACE2 group (P0.05). The results of Rac1 and Gem were consistent with the results of Western blot. The expression of CTGF, alpha -SMA and N-Cadherin were evaluated by Western blot technique. The results showed that the expression of three in AF-Control and AF-EGFP groups increased significantly, while the overexpression of ACE2 reversed this trend. The immunofluorescence method was used to detect the immunofluorescence. The expression of alpha -SMA showed that alpha -SMA was mainly distributed in cytoplasm. A large number of green fluorescent cells were found in group AF-Control and AF-EGFP, and the expression of alpha -SMA was stronger than that of Sham and AF-ACE2 groups. The results of Realtime RT-PCR showed c-Jun, c-myc and c-fos genes were expressed in the group of c-Jun, c-myc and c-fos. And AF-EGFP group significantly increased, and over expression of ACE2 in group AF-ACE2 could reduce the m RNA expression of three. Conclusion: (1) overexpression of ACE2 can reduce the induced rate of atrial fibrillation and shorten the duration of atrial fibrillation; (2) over expression of ACE2 regulates RAS imbalance caused by rapid atrial pacing, significantly downregulates the expression of AT1R, up AT2R, Ape The expression of Lin and Aplnr; (3) over expression of ACE2 can regulate the dynamic balance of Rac1 and Rad GTPase, significantly down regulation of the expression of Rac1 and up regulation of Rad GTPase, the expression of Gem, inhibition of fibrosis related factor CTGF, alpha -SMA and expression and down regulation and gene expression, in order to improve the remodeling of atrial fibrosis. In summary, this study The results showed that the over expression of ACE2 in the atrium was able to move the unbalanced RAS axially by regulating the atrial rapid pacing, up the expression of Rad GTPase and down regulation of the expression of Rac1 and inhibiting the expression of fibrosis related factors to improve atrial remodeling and the purpose of improving the atrial matrix, thus effectively reducing the room. The rate of fibrillation induction and the duration of atrial fibrillation are reduced, and the prevention and cure of atrial fibrillation is the ultimate benefit.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R541.75

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