本文選題：心肌肥厚 + 心力衰竭　； 參考：《東南大學(xué)》2017年博士論文

【摘要】：目的:心肌肥厚是一個多分子、多信號通路參與的復(fù)雜病理生理過程,可能導(dǎo)致心力衰竭、猝死等嚴重心血管事件,但是目前對于心肌肥厚發(fā)生發(fā)展的分子機制的認識仍十分有限。G蛋白信號通路調(diào)節(jié)蛋白(RGS)家族多個成員被發(fā)現(xiàn)參與心肌肥厚的病理生理過程。RGS12是分子量最大、功能結(jié)構(gòu)域最多的RGS家族成員。結(jié)構(gòu)的復(fù)雜性決定了 RGS12可以發(fā)揮更復(fù)雜和更精密的分子調(diào)控作用。本研究旨在闡明RGS12對心肌肥厚的影響及其具體機制。方法:第一部分:選取野生型(WT)小鼠及Sprague-Dawley大鼠乳鼠心肌細胞作為研究對象,分別采用胸主動脈縮窄術(shù)(AB)和血管緊張素Ⅱ (Ang Ⅱ)處理建立在體心肌肥厚和離體心肌細胞肥大模型,運用Western blot檢測造模后各時間點心肌組織、心肌細胞和成纖維細胞內(nèi)RGS12表達水平。第二部分:選取全系統(tǒng)RGS12基因敲除(RGS12-KO)小鼠和WT小鼠作為研究對象,采用AB構(gòu)建心肌肥厚模型。手術(shù)4周后檢測相關(guān)指標:取材小鼠心臟、肺臟和左側(cè)脛骨,計算心體比(HW/BW)、心脛比(HW/TL)和肺體比(LW/BW);采用HE、WGA、PSR等病理染色評價心肌細胞橫截面積及膠原沉積水平;超聲成像儀檢測小鼠心臟功能;應(yīng)用實時定量PCR檢測心肌肥厚和纖維化標志物水平。第三部分:選取心臟特異性RGS12轉(zhuǎn)基因(RGS12-CTG)小鼠和CAG-(loxP)CAT(loxP)-RGSl2/α-MHC-MerCreMer 雙轉(zhuǎn)基因(CRMC)小鼠作為研究對象,采用AB構(gòu)建心肌肥厚模型。手術(shù)4周后檢測相關(guān)指標:取材小鼠心臟、肺臟和左側(cè)脛骨,計算HW/BW、HW/TL和LW/BW;采用HE、WGA、PSR等病理染色評價心肌細胞橫截面積及膠原沉積水平;超聲成像儀檢測小鼠心臟功能;應(yīng)用實時定量PCR檢測心肌肥厚和纖維化標志物水平。第四部分:利用腺病毒轉(zhuǎn)染手段,構(gòu)建RGS12低表達(AdshRGS12)或高表達(AdRGS12)乳鼠心肌細胞,以腺病毒載體AdshRNA和AdGFP轉(zhuǎn)染乳鼠心肌細胞作為相應(yīng)的對照組,分別運用Ang Ⅱ和內(nèi)皮素(ET-1)構(gòu)建離體心肌細胞肥大模型。促肥厚因子干預(yù)48小時后檢測相關(guān)指標:采用免疫熒光染色比較測量心肌細胞表面積;應(yīng)用實時定量PCR檢測心肌肥厚標志物水平。第五部分:分別在動物實驗和細胞實驗中,運用Western blot檢測各組小鼠心臟組織或心肌細胞中MAPK信號通路分子的總量和磷酸化水平變化。然后,以Sprague-Dawley大鼠乳鼠心肌細胞為研究對象,采用Ang Ⅱ干預(yù)構(gòu)建離體心肌細胞肥大模型,運用免疫共沉淀檢測RGS12與信號通路關(guān)鍵分子的相互作用。最后,以RGS12-TG和CRMC小鼠作為研究對象,采用AB構(gòu)建心肌肥厚模型,使用信號通路分子特異性抑制干預(yù)小鼠。手術(shù)4周后檢測相關(guān)指標:取材小鼠心臟、肺臟和左側(cè)脛骨,計算HW/BW、HW/TL和LW/BW;采用HE、WGA、PSR等病理染色評價心肌細胞橫截面積及心肌纖維化程度;超聲成像儀檢測小鼠心臟功能。結(jié)果:第一部分:在體心肌肥厚模型和離體心肌細胞肥大模型中,心肌肥厚標志物表達水平明顯升高。與假手術(shù)組(Sham)相比,AB組小鼠心肌組織內(nèi)RGS12蛋白表達量明顯提高,且手術(shù)8周后的表達水平高于手術(shù)4周后。與在體實驗類似,Ang Ⅱ干預(yù)24小時和48小時后,心肌細胞內(nèi)RGS12的蛋白質(zhì)水平逐漸升高。然而,Ang Ⅱ干預(yù)后,成纖維細胞內(nèi)RGS12蛋白水平無明顯變化。第二部分:WT和RGS12-KO小鼠行假手術(shù)造模后,兩組小鼠的心臟大小、纖維化程度、心臟功能及相關(guān)基因表達水平無明顯差異。AB構(gòu)建心肌肥厚模型4周后,取材結(jié)果發(fā)現(xiàn),RGS12-KO小鼠的HW/BW、HW/TL和LW/BW小于WT小鼠;病理染色結(jié)果發(fā)現(xiàn),RGS12-KO小鼠的心臟大小、心肌細胞橫截面積、間質(zhì)和血管周圍膠原沉積以及膠原容積低于WT小鼠;心臟超聲結(jié)果發(fā)現(xiàn),RGS12-KO小鼠的左心腔擴大和左心收縮功能減低程度好于WT小鼠;RT-PCR檢測結(jié)果發(fā)現(xiàn),RGS12-KO小鼠的心肌肥厚標志物的mRNA表達水平低于WT小鼠。第三部分:CRMC和RGS12-CTG小鼠行假手術(shù)造模后,兩組小鼠的心臟大小、纖維化程度、心臟功能及相關(guān)基因表達水平無明顯差異。AB構(gòu)建心肌肥厚模型4周后,取材結(jié)果發(fā)現(xiàn),RGS12-CTG小鼠的HW/BW、HW/TL和LW/BW高于CRMC小鼠;病理染色結(jié)果發(fā)現(xiàn),RGS12-CTG小鼠的心臟大小、心肌細胞橫截面積、間質(zhì)和血管周圍膠原沉積以及膠原容積大于CRMC小鼠;心臟超聲結(jié)果發(fā)現(xiàn),RGS12-CTG小鼠的左心腔擴大和左心收縮功能減低程度較CRMC小鼠加重;RT-PCR檢測結(jié)果發(fā)現(xiàn),RGS12-CTG小鼠的心肌肥厚標志物的mRNA表達水平高于CRMC小鼠。第四部分:PBS 干預(yù) AdshRGS12、AdshRNA、AdRGS12 和 AdGFP 組乳鼠心肌細胞48小時后,各組乳鼠心肌細胞表面積和相關(guān)基因表達水平無明顯差異。Ang Ⅱ干預(yù)48小時后,免疫熒光染色結(jié)果發(fā)現(xiàn),AdshRGS12組心肌細胞表面積小于AdshRNA組,而AdRGS12組心肌細胞表面積大于AdGFP組;RT-PCR檢測結(jié)果發(fā)現(xiàn),AdshRGS12組心肌肥厚標志物的mRNA表達水平低于AdshRNA組,而AdRGS12組心肌肥厚標志物mRNA的表達水平高于AdGFP組。第五部分:促肥厚刺激不影響絲裂原活化蛋白激酶(MAPK)信號通路分子的蛋白質(zhì)總量,卻引起MAPK信號通路分子的磷酸化程度升高。其中,JNK1/2和P38的磷酸化程度不受RGS12表達水平的影響,而RGS12高表達提高MEK1/2和ERK1/2的磷酸化水平,RGS12低表達降低MEK1/2和ERK1/2的磷酸化水平。同時,Ang Ⅱ干預(yù)后,GS12能夠在心肌細胞內(nèi)與MEK1/2結(jié)合形成功能復(fù)合體。在RGS-CTG和CRMC小鼠心肌肥厚模型中,MEK1/2特異性抑制劑(U0126)干預(yù)能夠顯著降低小鼠的HW/BW、HW/TL和LW/BW,改善小鼠心肌肥厚、心肌細胞橫截面積增大、間質(zhì)和血管周圍膠原沉積,緩解左心腔擴大和左心收縮功能減低。更為重要的是,這些心臟肥厚性重構(gòu)指標在RGS-CTG/U0126和CRMC/U0126小鼠之間無明顯統(tǒng)計學(xué)差異。結(jié)論:本研究結(jié)果表明促肥厚刺激誘導(dǎo)心肌細胞內(nèi)RGS12表達升高,高表達的RGS12與MEK1/2形成功能復(fù)合體,促進MEK1/2-ERK1/2信號通路激活,促進壓力負荷誘導(dǎo)的心肌肥厚發(fā)生發(fā)展。因此,RGS12具有成為臨床治療心肌肥厚新靶點的潛力。
[Abstract]:Objective: myocardial hypertrophy is a complex pathophysiological process involving multiple molecules and multiple signal pathways, which may lead to severe cardiovascular events such as heart failure and sudden death. However, the understanding of the molecular mechanism of the development of cardiac hypertrophy is still limited, and many members of the.G protein signaling family (RGS) family have been found to be involved. The pathophysiological process of muscle hypertrophy (.RGS12) is the largest molecular weight and most functional domain of RGS family. The complexity of the structure determines that RGS12 can play a more complex and more sophisticated molecular regulation. This study aims to elucidate the effect of RGS12 on myocardial hypertrophy and its specific mechanisms. The cardiac myocytes of Sprague-Dawley rats were used as the research object. The models of cardiac hypertrophy and isolated myocardial hypertrophy were established by thoracic aorta coarctation (AB) and angiotensin II (Ang II) respectively. Western blot was used to detect the myocardium, myocardial cells and RGS12 expression of water in the fibroblasts. Second: the second part: select the whole system RGS12 gene knockout (RGS12-KO) mice and WT mice as the research object, construct the myocardial hypertrophy model with AB. After 4 weeks, the related indexes were measured: the heart, the lung and the left tibia were measured, the heart body ratio (HW/BW), the ratio of the tibia (HW/TL) and the lung body ratio (LW/BW) were calculated, and the pathological staining was evaluated by HE, WGA, PSR, etc. The transversal area of cardiac myocytes and the level of collagen deposition; the echocardiography was used to detect the cardiac function of mice; the level of myocardial hypertrophy and fibrosis markers was detected by real-time quantitative PCR. The third part: selected cardiac specific RGS12 transgenic (RGS12-CTG) mice and CAG- (loxP) CAT (loxP) -RGSl2/ a -MHC-MerCreMer double transgenic (CRMC) mice as research The model of cardiac hypertrophy was constructed with AB. After 4 weeks of operation, the related indexes were measured: HW/BW, HW/TL and LW/BW were calculated from the heart, lung and left tibia of mice. The myocardial cell cross section and collagen deposition were evaluated by HE, WGA, PSR and other pathological staining. The cardiac function of mice was detected by the ultrasonic imaging instrument, and the real-time quantitative PCR was used to detect the myocardium. The level of markers of hypertrophy and fibrosis. The fourth part: using adenovirus transfection to construct RGS12 low expression (AdshRGS12) or high expression (AdRGS12) rat myocardial cells, transfection of mammary rat cardiomyocytes with adenovirus vector AdshRNA and AdGFP as the corresponding control group, and use Ang II and endothelin (ET-1) to construct the large model of the isolated cardiomyocytes. Type. After 48 hours of hypertrophy intervention, the related indexes were measured: the surface area of cardiac myocytes was measured by immunofluorescence staining, and the level of myocardial hypertrophy markers was detected by real-time quantitative PCR. The fifth part: in animal experiment and cell experiment, the MAPK signal in heart tissues or cardiac myocytes of each group was detected by Western blot, respectively. The total amount of pathway molecules and the changes in the level of phosphorylation. Then, the rat cardiac myocytes of Sprague-Dawley rats were taken as the research object. Ang II intervention was used to construct the hypertrophy model of isolated cardiomyocytes, and the interaction between RGS12 and the key molecules of the signal pathway was detected by immunoprecipitation. Finally, the RGS12-TG and CRMC mice were used as the research object and AB was used. The model of myocardial hypertrophy was constructed, and the mice were treated with signal pathway molecular specific inhibition. After 4 weeks of operation, the related indexes were measured. HW/BW, HW/TL and LW/BW were calculated from the heart, lung and left tibia of mice. HE, WGA, PSR and other pathological staining were used to evaluate the cross sectional area of myocardial cells and the degree of myocardial fibrosis; the cardiac work of mice was detected by the ultrasonic imaging instrument. Results: the first part: the expression level of the markers of cardiac hypertrophy was significantly increased in the model of body hypertrophy and the hypertrophy of isolated cardiomyocytes. Compared with the sham operation group (Sham), the expression of RGS12 protein in the myocardium of the AB group was significantly increased, and the level of the expression was higher than that after the operation for 8 weeks after 4 weeks. It was similar to that in the body experiment, Ang II. After 24 hours and 48 hours of intervention, the protein level of RGS12 in myocardial cells increased gradually. However, there was no obvious change in the level of RGS12 protein in the fibroblasts after Ang II. Second: after the model of WT and RGS12-KO mice, the size of heart, the degree of fibroid, cardiac function and related gene expression level were not clear in the two groups of mice. 4 weeks after the significant difference of.AB, the results showed that HW/BW, HW/TL and LW/BW in RGS12-KO mice were less than WT mice. Pathological staining showed that the heart size, cross sectional area of cardiac myocytes, interstitial and perivascular collagen deposition and collagen volume of RGS12-KO mice were lower than that of WT mice; cardiac ultrasound findings found RGS12-KO mice The extent of left ventricular enlargement and left cardiac contractile function decreased better than that of WT mice. The results of RT-PCR detection showed that the mRNA expression level of the markers of myocardial hypertrophy in RGS12-KO mice was lower than that of WT mice. The third part: the size, degree of fibrosis, cardiac function and related gene expression in the two groups of mice after the model of CRMC and RGS12-CTG in the model of sham operation. 4 weeks after.AB construction of the myocardial hypertrophy model, the results showed that HW/BW, HW/TL and LW/BW in RGS12-CTG mice were higher than those of CRMC mice. Pathological staining showed that the heart size, cross section area of cardiac myocytes, interstitial and perivascular collagen deposition and collagen volume in RGS12-CTG mice were greater than that of CRMC mice; the echocardiographic results were more than that of CRMC mice. The left heart cavity enlargement and left cardiac contractile function of RGS12-CTG mice were worse than that of CRMC mice. The results of RT-PCR detection showed that the mRNA expression level of myocardial hypertrophy markers in RGS12-CTG mice was higher than that of CRMC mice. The fourth part: PBS intervention AdshRGS12, AdshRNA, AdRGS12 and AdGFP group of milk rat heart cells after 48 hours, each group of milk rat hearts There was no significant difference between the surface area of muscle cells and the expression level of related genes. After 48 hours of.Ang II intervention, the results of immunofluorescence staining found that the surface area of myocardial cells in group AdshRGS12 was less than that of group AdshRNA, and the surface area of cardiac myocytes in group AdRGS12 was larger than that in group AdGFP, and the results of RT-PCR detection found that the mRNA expression level of myocardial hypertrophy markers in group AdshRGS12 was lower than that of group AdGFP. The expression level of mRNA, a marker of myocardial hypertrophy in group AdRGS12, is higher than that in group AdGFP. Fifth: the fifth part: hypertrophy stimulation does not affect the total protein of the mitogen activated protein kinase (MAPK) signaling pathway molecules, but increases the phosphorylation of MAPK signaling molecules. In this, the degree of phosphorylation of JNK1/2 and P38 is not affected by RGS12 expression. The high expression of RGS12 increases the phosphorylation level of MEK1/2 and ERK1/2, and the low expression of RGS12 reduces the phosphorylation level of MEK1/2 and ERK1/2. At the same time, GS12 can combine with MEK1/2 in cardiac myocytes to form functional complexes. In RGS-CTG and CRMC murine cardiac hypertrophy models, MEK1/2 specific inhibitors can intervene. Significantly reduced HW/BW, HW/TL and LW/BW in mice, improved myocardial hypertrophy in mice, increased cross sectional area of cardiac myocytes, interstitial and perivascular collagen deposition, alleviated left ventricular enlargement and left cardiac contractile dysfunction. More importantly, these cardiac hypertrophy indices were not statistically poor between RGS-CTG/U0126 and CRMC/U0126 mice. Conclusion: the results of this study showed that hypertrophy induced the increase of RGS12 expression in cardiac myocytes, high expression of RGS12 and MEK1/2 to form functional complexes, promote the activation of MEK1/2-ERK1/2 signaling pathway and promote the development of myocardial hypertrophy induced by pressure load. Therefore, RGS12 has the potential to be a new target for clinical treatment of myocardial hypertrophy.

【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R542.2

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