【摘要】：目的：心力衰竭是各種終末期心臟疾病的最終歸宿,其發(fā)病率與病死率高,嚴(yán)重危害人類健康。在各種心血管疾病,如高血壓,心肌梗死和心臟瓣膜疾病等因素刺激下,心肌細(xì)胞會(huì)代償性肥大以適應(yīng)泵血的需要,然而心肌細(xì)胞病理性肥大所導(dǎo)致的心肌肥厚卻是心力衰竭發(fā)生發(fā)展最重要的獨(dú)立危險(xiǎn)因素。由于心肌肥厚發(fā)病機(jī)制目前尚未充分探明,因而目前仍然缺乏有效治療靶點(diǎn)及治療藥物。近二十年來(lái),鈣離子反應(yīng)性蛋白分子Calcineurin的過(guò)度激活被認(rèn)為是導(dǎo)致心肌細(xì)胞肥大及心肌肥厚最為重要的細(xì)胞內(nèi)分子機(jī)制之一,然而關(guān)于其負(fù)調(diào)控機(jī)制卻研究甚少。EPI64C蛋白分子是在T細(xì)胞中被發(fā)現(xiàn)的一種Calcineurin與Ras信號(hào)通路雙重抑制蛋白,并且在T細(xì)胞增殖及活化中起著重要的負(fù)調(diào)控作用,然而關(guān)于其在心臟中的作用卻不清楚。本研究旨在闡明EPI64C在病理性心肌肥厚中的作用及其機(jī)制,并探索其在治療心肌肥厚疾病中的轉(zhuǎn)化應(yīng)用價(jià)值。方法：1.建立主動(dòng)脈縮窄誘導(dǎo)的小鼠心肌肥厚模型,觀察EPI64C在壓力過(guò)負(fù)荷誘導(dǎo)的小鼠心肌肥厚過(guò)程中心肌表達(dá)水平的改變情況；收集擴(kuò)張型心肌病及肥厚型心肌病病人心臟移植術(shù)后的左室心肌樣本,并以正常心臟左室心肌樣本為對(duì)照,探明EPI64C在心肌正常及病理狀態(tài)下的表達(dá)情況。2.建立Ang Ⅱ刺激誘導(dǎo)新生大鼠心肌細(xì)胞肥大的體外模型,檢測(cè)EPI64C在肥大心肌細(xì)胞中的表達(dá)變化情況；使用慢病毒和腺病毒載體分別沉默和過(guò)表達(dá)EPI64C,觀察EPI64C功能增強(qiáng)或功能缺失對(duì)心肌細(xì)胞肥大的影響。3.構(gòu)建心肌細(xì)胞特異性的Epi64c基因敲除小鼠,和心肌細(xì)胞特異性的Epi64c轉(zhuǎn)基因小鼠,檢驗(yàn)EPI64C功能缺失和功能增強(qiáng)對(duì)小鼠壓力過(guò)負(fù)荷誘導(dǎo)的心肌肥厚的作用。4.構(gòu)建Ras抑制功能缺失的突變EPI64C轉(zhuǎn)基因小鼠,探尋EPI64C調(diào)控心肌肥厚是否通過(guò)抑制Ras活性發(fā)揮作用。5.構(gòu)建Calcineurin結(jié)合功能缺失的截短EPI64C轉(zhuǎn)基因小鼠,解析EPI64C調(diào)控心肌肥厚是否通過(guò)抑制Calcineurin活性發(fā)揮作用,并闡明其具體作用機(jī)制。6.構(gòu)建過(guò)表達(dá)EPI64C的慢病毒載體,體內(nèi)轉(zhuǎn)染食蟹猴心臟。建立猴主動(dòng)脈縮窄誘導(dǎo)的心肌肥厚模型,探索過(guò)表達(dá)EPI64C的治療策略對(duì)猴心肌肥厚的治療和預(yù)防作用。結(jié)果：1.相比于正常心臟組織中較高的表達(dá)水平,EPI64C隨著小鼠心肌肥厚的發(fā)展逐漸下降,并且在人擴(kuò)張型心肌病及肥厚型心肌病左室心肌中表達(dá)量明顯下降。2.體外敲低或過(guò)表達(dá)EPI64C表達(dá)水平可以分別顯著加重或減輕Ang Ⅱ誘導(dǎo)的新生大鼠心肌細(xì)胞肥大。3.心肌細(xì)胞中Epi64c被敲除后,主動(dòng)脈縮窄所誘導(dǎo)的心肌肥厚顯著加重；反之,EPI64C在心肌細(xì)胞中過(guò)表達(dá)可以抑制主動(dòng)脈縮窄誘導(dǎo)的心肌肥厚。4.EPI64C并不通過(guò)抑制Ras的活性發(fā)揮對(duì)心肌肥厚的負(fù)調(diào)控作用。5. EPI64C抑制心肌肥厚的機(jī)制在于直接結(jié)合到Calcineurin的酶活性核心區(qū)域并抑制Calcineurin的活性,進(jìn)而抑制下游NFAT轉(zhuǎn)錄活性。6.猴心臟過(guò)表達(dá)EPI64C可以顯著抑制主動(dòng)脈縮窄誘導(dǎo)的心肌肥厚。結(jié)論：EPI64C通過(guò)抑制Calcineurin-NFAT信號(hào)通路抑制壓力過(guò)負(fù)荷誘導(dǎo)的心肌肥厚,是病理性心肌肥厚不可或缺的負(fù)調(diào)控蛋白,其表達(dá)水平降低與病理性心肌肥厚的發(fā)生發(fā)展有關(guān)。過(guò)表達(dá)EPI64C的基因治療策略在猴壓力過(guò)負(fù)荷誘導(dǎo)的心肌肥厚中的成功治療應(yīng)用,提示EPI64C可以作為潛在的心肌肥厚治療靶標(biāo),有重要的臨床轉(zhuǎn)化應(yīng)用價(jià)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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R542.2;R541.6

【共引文獻(xiàn)】

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