EPI64C在病理性心肌肥厚中的作用，机制及转化应用研究
发布时间：2018-08-16 13:06
【摘要】：目的：心力衰竭是各种终末期心脏疾病的最终归宿,其发病率与病死率高,严重危害人类健康。在各种心血管疾病,如高血压,心肌梗死和心脏瓣膜疾病等因素刺激下,心肌细胞会代偿性肥大以适应泵血的需要,然而心肌细胞病理性肥大所导致的心肌肥厚却是心力衰竭发生发展最重要的独立危险因素。由于心肌肥厚发病机制目前尚未充分探明,因而目前仍然缺乏有效治疗靶点及治疗药物。近二十年来,钙离子反应性蛋白分子Calcineurin的过度激活被认为是导致心肌细胞肥大及心肌肥厚最为重要的细胞内分子机制之一,然而关于其负调控机制却研究甚少。EPI64C蛋白分子是在T细胞中被发现的一种Calcineurin与Ras信号通路双重抑制蛋白,并且在T细胞增殖及活化中起着重要的负调控作用,然而关于其在心脏中的作用却不清楚。本研究旨在阐明EPI64C在病理性心肌肥厚中的作用及其机制,并探索其在治疗心肌肥厚疾病中的转化应用价值。方法：1.建立主动脉缩窄诱导的小鼠心肌肥厚模型,观察EPI64C在压力过负荷诱导的小鼠心肌肥厚过程中心肌表达水平的改变情况；收集扩张型心肌病及肥厚型心肌病病人心脏移植术后的左室心肌样本,并以正常心脏左室心肌样本为对照,探明EPI64C在心肌正常及病理状态下的表达情况。2.建立Ang Ⅱ刺激诱导新生大鼠心肌细胞肥大的体外模型,检测EPI64C在肥大心肌细胞中的表达变化情况；使用慢病毒和腺病毒载体分别沉默和过表达EPI64C,观察EPI64C功能增强或功能缺失对心肌细胞肥大的影响。3.构建心肌细胞特异性的Epi64c基因敲除小鼠,和心肌细胞特异性的Epi64c转基因小鼠,检验EPI64C功能缺失和功能增强对小鼠压力过负荷诱导的心肌肥厚的作用。4.构建Ras抑制功能缺失的突变EPI64C转基因小鼠,探寻EPI64C调控心肌肥厚是否通过抑制Ras活性发挥作用。5.构建Calcineurin结合功能缺失的截短EPI64C转基因小鼠,解析EPI64C调控心肌肥厚是否通过抑制Calcineurin活性发挥作用,并阐明其具体作用机制。6.构建过表达EPI64C的慢病毒载体,体内转染食蟹猴心脏。建立猴主动脉缩窄诱导的心肌肥厚模型,探索过表达EPI64C的治疗策略对猴心肌肥厚的治疗和预防作用。结果：1.相比于正常心脏组织中较高的表达水平,EPI64C随着小鼠心肌肥厚的发展逐渐下降,并且在人扩张型心肌病及肥厚型心肌病左室心肌中表达量明显下降。2.体外敲低或过表达EPI64C表达水平可以分别显著加重或减轻Ang Ⅱ诱导的新生大鼠心肌细胞肥大。3.心肌细胞中Epi64c被敲除后,主动脉缩窄所诱导的心肌肥厚显著加重；反之,EPI64C在心肌细胞中过表达可以抑制主动脉缩窄诱导的心肌肥厚。4.EPI64C并不通过抑制Ras的活性发挥对心肌肥厚的负调控作用。5. EPI64C抑制心肌肥厚的机制在于直接结合到Calcineurin的酶活性核心区域并抑制Calcineurin的活性,进而抑制下游NFAT转录活性。6.猴心脏过表达EPI64C可以显著抑制主动脉缩窄诱导的心肌肥厚。结论：EPI64C通过抑制Calcineurin-NFAT信号通路抑制压力过负荷诱导的心肌肥厚,是病理性心肌肥厚不可或缺的负调控蛋白,其表达水平降低与病理性心肌肥厚的发生发展有关。过表达EPI64C的基因治疗策略在猴压力过负荷诱导的心肌肥厚中的成功治疗应用,提示EPI64C可以作为潜在的心肌肥厚治疗靶标,有重要的临床转化应用价值。
[Abstract]:Objective: Heart failure is the ultimate outcome of various end-stage heart diseases with high morbidity and mortality, which seriously endangers human health. Under the stimulation of various cardiovascular diseases, such as hypertension, myocardial infarction and heart valve disease, cardiomyocytes will be compensatory hypertrophy to meet the needs of blood pumping, but cardiomyocytes pathological hypertrophy. Myocardial hypertrophy is the most important independent risk factor for the development of heart failure. As the pathogenesis of myocardial hypertrophy has not been fully explored, there is still a lack of effective therapeutic targets and drugs. EPI64C protein is a double inhibitor of Calcineurin and Ras signaling pathway found in T cells, and plays an important negative role in T cell proliferation and activation. However, there is little research on its negative regulatory mechanism. The purpose of this study was to elucidate the role of EPI64C in pathological myocardial hypertrophy and its mechanism, and to explore its application value in the treatment of myocardial hypertrophy. Methods: 1. Establish a mouse model of myocardial hypertrophy induced by aortic coarctation and observe the process of myocardial hypertrophy induced by pressure overload. The expression of EPI64C in normal and pathological myocardium was detected by collecting left ventricular myocardium samples from patients with dilated cardiomyopathy and hypertrophic cardiomyopathy after cardiac transplantation, and comparing with normal left ventricular myocardium samples. 2. Ang II stimulation was established to induce cardiomyocyte hypertrophy in neonatal rats. In vitro model, the expression of EPI64C in hypertrophic cardiomyocytes was detected. Lentiviral and adenoviral vectors were used to silence and overexpress EPI64C respectively to observe the effect of enhanced or dysfunctional EPI64C on cardiomyocyte hypertrophy. 3. The cardiomyocyte-specific Epi64c knockout mice and cardiomyocyte-specific Epi64c were constructed. Transgenic mice were used to examine the effects of EPI64C deficiency and enhancement on myocardial hypertrophy induced by pressure overload in mice. 4. Ras-inhibiting mutant EPI64C transgenic mice were constructed to explore whether EPI64C could regulate myocardial hypertrophy by inhibiting Ras activity. 5. Calcineurin-binding deleted truncated EPI64C transgene was constructed. To investigate whether EPI64C plays a role in myocardial hypertrophy by inhibiting Calcineurin activity in mice and elucidate its specific mechanism. 6. Lentiviral vector overexpressing EPI64C was constructed to transfect the heart of cynomolgus monkeys in vivo. Results: 1. Compared with the high expression level in normal heart tissue, EPI64C gradually decreased with the development of myocardial hypertrophy in mice, and the expression level of EPI64C in left ventricular myocardium of human dilated cardiomyopathy and hypertrophic cardiomyopathy decreased significantly. Mild Ang II-induced cardiomyocyte hypertrophy in neonatal rats. 3. Epi64c in cardiomyocytes was knocked out, cardiomyocyte hypertrophy induced by aortic coarctation was significantly aggravated; conversely, the overexpression of EPI64C in cardiomyocytes could inhibit cardiomyocyte hypertrophy induced by aortic coarctation. 4. EPI64C did not exert a negative regulation of cardiomyocyte hypertrophy by inhibiting the activity of Ras. 5. EPI64C inhibits cardiac hypertrophy by binding directly to the core of Calcineurin enzyme activity and inhibiting Calcineurin activity, thereby inhibiting downstream NFAT transcriptional activity. 6. Overexpression of EPI64C in monkey heart can significantly inhibit cardiac hypertrophy induced by aortic coarctation. Pathway inhibition of pressure overload-induced cardiac hypertrophy is an indispensable negative regulatory protein in pathological myocardial hypertrophy. The decrease of its expression level is related to the occurrence and development of pathological myocardial hypertrophy. Potential therapeutic targets for cardiac hypertrophy have important clinical application value.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R542.2;R541.6
本文编号：2186069
[Abstract]:Objective: Heart failure is the ultimate outcome of various end-stage heart diseases with high morbidity and mortality, which seriously endangers human health. Under the stimulation of various cardiovascular diseases, such as hypertension, myocardial infarction and heart valve disease, cardiomyocytes will be compensatory hypertrophy to meet the needs of blood pumping, but cardiomyocytes pathological hypertrophy. Myocardial hypertrophy is the most important independent risk factor for the development of heart failure. As the pathogenesis of myocardial hypertrophy has not been fully explored, there is still a lack of effective therapeutic targets and drugs. EPI64C protein is a double inhibitor of Calcineurin and Ras signaling pathway found in T cells, and plays an important negative role in T cell proliferation and activation. However, there is little research on its negative regulatory mechanism. The purpose of this study was to elucidate the role of EPI64C in pathological myocardial hypertrophy and its mechanism, and to explore its application value in the treatment of myocardial hypertrophy. Methods: 1. Establish a mouse model of myocardial hypertrophy induced by aortic coarctation and observe the process of myocardial hypertrophy induced by pressure overload. The expression of EPI64C in normal and pathological myocardium was detected by collecting left ventricular myocardium samples from patients with dilated cardiomyopathy and hypertrophic cardiomyopathy after cardiac transplantation, and comparing with normal left ventricular myocardium samples. 2. Ang II stimulation was established to induce cardiomyocyte hypertrophy in neonatal rats. In vitro model, the expression of EPI64C in hypertrophic cardiomyocytes was detected. Lentiviral and adenoviral vectors were used to silence and overexpress EPI64C respectively to observe the effect of enhanced or dysfunctional EPI64C on cardiomyocyte hypertrophy. 3. The cardiomyocyte-specific Epi64c knockout mice and cardiomyocyte-specific Epi64c were constructed. Transgenic mice were used to examine the effects of EPI64C deficiency and enhancement on myocardial hypertrophy induced by pressure overload in mice. 4. Ras-inhibiting mutant EPI64C transgenic mice were constructed to explore whether EPI64C could regulate myocardial hypertrophy by inhibiting Ras activity. 5. Calcineurin-binding deleted truncated EPI64C transgene was constructed. To investigate whether EPI64C plays a role in myocardial hypertrophy by inhibiting Calcineurin activity in mice and elucidate its specific mechanism. 6. Lentiviral vector overexpressing EPI64C was constructed to transfect the heart of cynomolgus monkeys in vivo. Results: 1. Compared with the high expression level in normal heart tissue, EPI64C gradually decreased with the development of myocardial hypertrophy in mice, and the expression level of EPI64C in left ventricular myocardium of human dilated cardiomyopathy and hypertrophic cardiomyopathy decreased significantly. Mild Ang II-induced cardiomyocyte hypertrophy in neonatal rats. 3. Epi64c in cardiomyocytes was knocked out, cardiomyocyte hypertrophy induced by aortic coarctation was significantly aggravated; conversely, the overexpression of EPI64C in cardiomyocytes could inhibit cardiomyocyte hypertrophy induced by aortic coarctation. 4. EPI64C did not exert a negative regulation of cardiomyocyte hypertrophy by inhibiting the activity of Ras. 5. EPI64C inhibits cardiac hypertrophy by binding directly to the core of Calcineurin enzyme activity and inhibiting Calcineurin activity, thereby inhibiting downstream NFAT transcriptional activity. 6. Overexpression of EPI64C in monkey heart can significantly inhibit cardiac hypertrophy induced by aortic coarctation. Pathway inhibition of pressure overload-induced cardiac hypertrophy is an indispensable negative regulatory protein in pathological myocardial hypertrophy. The decrease of its expression level is related to the occurrence and development of pathological myocardial hypertrophy. Potential therapeutic targets for cardiac hypertrophy have important clinical application value.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R542.2;R541.6
【共引文献】
相关期刊论文 前2条
1 张德明;;探究性学习在初中数学课堂中的尝试[J];数学大世界(上旬);2017年01期
2 陈慧香;;初中数学探究型课堂的探索——关于探究活动设计问题[J];新课程(上);2014年03期
相关硕士学位论文 前10条
1 邓享裕;初中数学探究性学习内容选择的适恰性研究[D];福建师范大学;2016年
2 吴梦园;引导发现法在小学数学概念教学中的运用研究[D];杭州师范大学;2016年
3 陈丽清;初中平面几何的概念课教学设计研究[D];四川师范大学;2016年
4 王力;新课程理念下中学探究性教学的实践探究[D];华中师范大学;2015年
5 王亚君;农村高中数学问题导学教学模式的构建与案例分析[D];河北师范大学;2015年
6 宋琳琳;中英初中数学教材中定义引入方式的比较研究[D];东北师范大学;2015年
7 郑彤阳;初中导学案应用现状、问题及对策研究[D];辽宁师范大学;2015年
8 刘蓉;高中数学学案导学教学模式的现状研究[D];首都师范大学;2014年
9 耿华;导学案在高中数学教学中的实践研究[D];西北师范大学;2014年
10 倪建娥;一体化教学案在高中必修化学教学中的实践研究[D];苏州大学;2013年
，本文编号：2186069
本文链接：https://www.wllwen.com/yixuelunwen/xxg/2186069.html
最近更新
教材专著
