本文選題：hHole + Pygopus1�。� 參考：《湖南師范大學(xué)》2016年博士論文

【摘要】：病理性心肌肥厚(Pathological Cardiac Hypertrophy,PCH)是臨床各類心臟疾病發(fā)展終末期的一個共同病理過程。據(jù)報道,該疾病在全球范圍內(nèi)的人群發(fā)病率約0.2%,在我國的成人群體中發(fā)病率約0.18%,約100萬患者,且呈逐年增長趨勢。PCH的發(fā)生、發(fā)展涉及多基因、多通路、多環(huán)節(jié)及多層次,迄今至少已發(fā)現(xiàn)有20個基因1400多種突變與PCH的發(fā)生相關(guān)。但仍有許多調(diào)控基因有待鑒定,其發(fā)生、發(fā)展的分子機制亦有待闡明。hHole與Pygo1均是在脊椎動物成體心臟組織中特異性高表達的基因,我們的前期研究結(jié)果顯示在人類心衰的心肌標(biāo)本中,hHole和Pygo1的均出現(xiàn)異常表達,然而對于hHole和Pygo1基因功能的研究,尤其是在心臟中的功能研究,迄今國際上尚未見相關(guān)報道。因此本文首先應(yīng)用生物信息學(xué)方法,對人HOLE及人PYGO1蛋白的基本理化性質(zhì)、結(jié)構(gòu)特征等進行預(yù)測分析,并進一步通過體內(nèi)遺傳修飾手段構(gòu)建心肌特異性過表達的轉(zhuǎn)基因動物模型,旨在通過建立hHole和Pygo1過表達轉(zhuǎn)基因小鼠模型,分別探討hHole和Pygo1在PCH發(fā)生中的作用,并進一步闡明其潛在的作用機理。一、心肌特異性過表達hHole阻遏ISO誘導(dǎo)的病理性心肌肥厚Hole基因作為一個在心臟發(fā)育和成體期均有高表達的基因,已經(jīng)被發(fā)現(xiàn)十多年,但國際同行們似乎一直忽視了它在體內(nèi)的生物學(xué)功能,Hole基因的生物學(xué)功能迄今仍是一片空白。我們的前期研究結(jié)果揭示,HOLE蛋白具有ERK結(jié)合位點(D-domain)和結(jié)合SH3結(jié)構(gòu)域的多聚脯氨酸序列(PXXP),且上述結(jié)構(gòu)域?qū)RF肥大信號具有一定的抑制作用。此外,有報道發(fā)現(xiàn)人類hHole基因(TMEM121)內(nèi)部PXXP位點的SNP與人類先天性心臟病存在一定的相關(guān)性,似乎使得hHole基因在心臟中的功能變得可以預(yù)知。在前期研究中,我們還發(fā)現(xiàn)在人類擴張型心肌病患者中,hHole mRNA表達顯著升高,進一步在體內(nèi)外水平分析心肌肥厚的小鼠模型及心肌細胞肥大的細胞模型中Hole的表達水平,發(fā)現(xiàn)Hole在mRNA和蛋白水平較對照組均有顯著升高,上述研究結(jié)果提示Hole可能參與病理性心肌肥厚的調(diào)控。為研究hHole基因在病理性心肌肥厚的發(fā)生、發(fā)展過程中的作用,我們首先應(yīng)用生物信息學(xué)方法對人類hHole基因的基本結(jié)構(gòu)、理化性質(zhì)等進行了預(yù)測分析,其次,為從實驗水平驗證人類hHole基因的相關(guān)功能,我們應(yīng)用體內(nèi)遺傳修飾手段,首次建立了心臟特異性hHole過表達轉(zhuǎn)基因小鼠模型。我們發(fā)現(xiàn)轉(zhuǎn)基因小鼠在正常的生理條件下,表型正常,但效應(yīng)于異丙腎上腺(ISO)刺激,轉(zhuǎn)基因的小鼠較WT小鼠表現(xiàn)出抵抗ISO誘發(fā)的心肌肥厚,具體表現(xiàn)為心肌細胞面積的細微變化、輕微的心肌纖維沉積、良好的心功能輸出等。上述結(jié)果初步證實hHole基因可能是PCH的抑制因子,我們的研究結(jié)果首次從分子水平上揭示hHole基因在脊椎動物成體內(nèi)的生物學(xué)功能。為深入研究hHole抑制病理性心肌肥厚的作用機理,我們進一步應(yīng)用q RT-PCR方法分析TG小鼠中MAPK家族成員在mRNA水平的表達變化,結(jié)果發(fā)現(xiàn)ISO刺激的轉(zhuǎn)基因小鼠中ERK1,ERK2的mRNA表達水平均有下調(diào),JNK或P38在mRNA水平則無顯著性變化。進一步應(yīng)用W B方法檢測轉(zhuǎn)基因小鼠心臟組織中p-ERK及t-ERK的表達變化,結(jié)果顯示:效應(yīng)于ISO刺激,心肌特異性過表達hHole可顯著抑制ERK1/2的磷酸化。上述研究結(jié)果提示hHole可能通過ERKs信號途經(jīng)在轉(zhuǎn)錄和翻譯后水平雙重調(diào)控病理性心肌肥厚。二、心肌特異性過表達Pygo1自主調(diào)控病理性心肌肥厚果蠅Pygopus基因是2002年發(fā)現(xiàn)的經(jīng)典WNT信號通路的關(guān)鍵成員。具有調(diào)控組織發(fā)育、染色質(zhì)重塑、轉(zhuǎn)錄激活等多種重要生物學(xué)功能。在脊椎動物中Pygopus有兩種同源基因,分別是Pygopus1和Pygopus2(Pygo1,Pygo2)。在成體小鼠中,Pygo2基因在多種組織中廣泛表達,但Pygo1僅在心臟組織中特異性高表達。目前對Pygo1的研究相對甚少,其在脊椎動物成體心臟中的功能研究更是一片空白。Pygo1基因在成體心臟中特異性高表達是否提示其可能與心臟功能密切相關(guān)?前期研究發(fā)現(xiàn)人類心肌肥厚患者中PGYO1蛋白表達異常升高,心肌肥厚小鼠模型中Pygo1在mRNA和蛋白表達與“胚胎基因”ANF、β-MHC、SK-α-actin的表達呈一致性上調(diào)。進一步在體外細胞水平的研究結(jié)果發(fā)現(xiàn)穩(wěn)定過表達Pygo1的細胞系無需其它病理性肥厚刺激因子,即表現(xiàn)出細胞體積的增大和肥厚“標(biāo)志基因”的上調(diào);而在干擾Pygo1表達的細胞系中,被干擾細胞系可抑制由ISO或胎牛血清誘導(dǎo)的心肌肥大。以上結(jié)果提示Pygo1基因可能是調(diào)控PCH發(fā)生、發(fā)展的一個關(guān)鍵性作用因子。為深入研究Pygo1的生物學(xué)功能,我們首先應(yīng)用生物信息學(xué)方法對人類Pygo1基因的基本結(jié)構(gòu)及理化性質(zhì)進行預(yù)測分析。其次,為進一步證實該基因在動物整體水平的表達效應(yīng),我們將Pgyo1基因構(gòu)建在α-MHC啟動子之下,并通過囊胚注射獲得了過表達Pygo1的轉(zhuǎn)基因小鼠后代,初步研究發(fā)現(xiàn)不需要任何肥大病理因子的刺激,Pygo1過表達成年小鼠中的“胚胎基因”ANP、β-MHC、SK-α-actin在mRNA水平的表達顯著性上調(diào),并伴有心肌細胞體積的增大及年齡相關(guān)的心肌纖維化等。我們的研究結(jié)果首次揭示Pygo1基因可自主調(diào)控PCH的發(fā)生,但其作用機理有待進一步的研究。病理性心肌肥厚的發(fā)生、發(fā)展機制非常復(fù)雜,我們首次應(yīng)用生物信息學(xué)方法預(yù)測分析人hHole及Pygo1基因的基本理化性質(zhì)及基本結(jié)構(gòu)等,并在動物整體水平驗證了Hole及Pygo1在小鼠成體心臟內(nèi)的生物學(xué)功能。發(fā)現(xiàn)hHole可通過ERK信號途徑在轉(zhuǎn)錄和翻譯后水平雙重抑制PCH的發(fā)生、發(fā)展。而心肌特異性過表達Pygo1可自主誘導(dǎo)PCH的發(fā)生。隨著研究的深入,PCH的靶點干預(yù)似有曙光。后續(xù)研究中進一步從分子水平研究其關(guān)鍵靶點及開發(fā)靶向性藥物,對于PCH的治療及心力衰竭的防治將具重要的理論和現(xiàn)實意義。
[Abstract]:Pathological Cardiac Hypertrophy (PCH) is a common pathological process in the end-stage of the development of various kinds of heart diseases. It is reported that the incidence of the disease is about 0.2% in the global population. The incidence of the disease is about 0.18% in the adult population in our country, about 1 million of the patients, and the incidence of.PCH is growing year by year. It involves multiple genes, multiple pathways, multilinks and multilevels. At least 1400 mutations have been found to be related to the occurrence of PCH in at least 20 genes. However, there are still many regulatory genes to be identified. The molecular mechanism of development and the molecular mechanism of development are also to be elucidated by both.HHole and Pygo1, which are highly expressed in the adult cardiac tissue of vertebrates. The previous study showed that the abnormal expression of hHole and Pygo1 in the heart failure of human heart failure, however, the study on the function of hHole and Pygo1, especially in the heart function, has not been reported in the world. Therefore, this paper first applies the method of bioinformatics to the base of human HOLE and human PYGO1 protein. The physical and chemical properties, structural characteristics and so on were predicted, and the transgenic animal model of myocardium specific overexpression was constructed by means of genetic modification. The purpose of this study was to explore the role of hHole and Pygo1 in the occurrence of PCH by establishing hHole and Pygo1 over expression transgenic mice and further elucidate the potential mechanisms. The Hole gene of myocardial hypertrophy induced by myocardial specific overexpression of hHole repression ISO as a gene that has high expression in heart development and adult stage has been found for more than 10 years, but the international counterparts seem to have neglected its biological function in the body, and the biological function of the Hole gene is still a blank. Our previous study revealed that HOLE protein has a ERK binding site (D-domain) and a polyproline sequence (PXXP) binding to the SH3 domain, and the above domain has a certain inhibitory effect on the SRF hypertrophy signal. In addition, it is reported that SNP of the PXXP loci of the human hHole gene (TMEM121) is present with human congenital heart disease. The definite correlation seems to make the function of the hHole gene predictable in the heart. In previous studies, we also found a significant increase in the expression of hHole mRNA in human dilated cardiomyopathy. The level of Hole expression in the model of cardiac hypertrophy and the cell model of cardiac myocyte hypertrophy was further analyzed in vitro and in vivo. It was found that Hole was significantly higher in mRNA and protein levels than in the control group. The above results suggest that Hole may be involved in the regulation of pathological myocardial hypertrophy. In order to study the role of hHole gene in the pathogenesis of pathological myocardial hypertrophy, we first apply the Bioinformatics Method to the basic structure and physicochemical properties of the human hHole gene. Secondly, in order to verify the related function of human hHole gene from the experimental level, we first established a transgenic mouse model with heart specific hHole overexpression by means of genetic modification. We found that the transgenic mice were normal under normal physiological conditions, but the effect was on the ISO spines. The transgenic mice showed the myocardial hypertrophy induced by ISO in the transgenic mice than in the WT mice, which was specific to the subtle changes in the area of the cardiac myocytes, the slight cardiac muscle deposition, and the good cardiac output. The results preliminarily confirmed that the hHole gene might be a inhibitory factor of PCH. Our results were the first to reveal the hHole from the molecular level. The biological function of the gene in the body of vertebrates. In order to further study the mechanism of hHole inhibition of pathological myocardial hypertrophy, we further applied the Q RT-PCR method to analyze the expression changes of MAPK family members at mRNA level in TG mice. The results showed that the expression level of ERK1 in ISO stimulated transgenic mice was down, JNK or P was reduced. 38 there was no significant change at mRNA level. W B method was further used to detect the expression of p-ERK and t-ERK in the cardiac tissue of transgenic mice. The results showed that the effect on ISO stimulation, cardiac specific overexpression hHole could significantly inhibit the phosphorylation of ERK1/2. The results suggested that hHole may pass through the ERKs signal through the transcription and translation. Level double regulation of pathological myocardial hypertrophy. Two, the Pygopus gene of cardiac hyperexpression Pygo1 is the key member of the classic WNT signaling pathway found in 2002. It has many important biological functions, such as regulation of tissue development, chromatin remodeling, transcription activation, and so on. There are two kinds of Pygopus in vertebrates. Homologous genes are Pygopus1 and Pygopus2 (Pygo1, Pygo2). In adult mice, the Pygo2 gene is widely expressed in a variety of tissues, but Pygo1 is only highly expressed in the heart tissue. The study of Pygo1 is relatively small. The study of the work ability in the adult heart of vertebrates is a blank.Pygo1 gene in the adult heart. Does the high expression of heterosexual expression suggest that it may be closely related to cardiac function? Earlier studies have found that the expression of PGYO1 protein in the human hypertrophy of human cardiac hypertrophy is abnormal, and the expression of Pygo1 in mRNA and protein expression in the murine model of myocardial hypertrophy is up regulated in accordance with the expression of "embryo gene" ANF, beta -MHC, SK- alpha -actin. The results showed that the cell lines that stably overexpressed Pygo1 did not need other pathological hypertrophic stimulating factors, that is, the increase of cell volume and the up-regulation of the hypertrophy "marker gene", and in the cell lines interfering with Pygo1 expression, the interfered cell lines could inhibit the myocardial hypertrophy induced by ISO or fetal bovine serum. The above results suggest the Pygo1 gene. It may be a key factor in regulating the development of PCH. In order to further study the biological function of Pygo1, we first applied bioinformatics to predict the basic structure and physicochemical properties of the human Pygo1 gene. Secondly, to further confirm the expression effect of the gene on the whole level of the animal, we will be based on the Pgyo1 base. Under the construction of the alpha -MHC promoter and injected through the blastocyst, the transgenic mice that overexpressed Pygo1 were obtained. The preliminary study found that no hypertrophic pathological factors were needed. Pygo1 overexpressed the expression of "embryo gene" ANP, beta -MHC, SK- alpha -actin in mRNA level in adult mice, and was accompanied by cardiac myocyte body. Our results first revealed that the Pygo1 gene can regulate the occurrence of PCH autonomously, but its mechanism needs further study. The pathogenesis of pathological myocardial hypertrophy is very complex. We first use bioinformatics methods to predict the basis of human hHole and Pygo1 gene. The biological function of Hole and Pygo1 in the adult heart of mice is verified by the physical and chemical properties and basic structures. It is found that hHole can inhibit the occurrence and development of PCH both at both transcriptional and post-translational levels through ERK signal pathway. And the myocardial specific overexpression Pygo1 can induce the occurrence of PCH autonomously. With the further research, P The target intervention of CH seems to be dawning. Further research on the key targets and the development of targeted drugs from the molecular level will be of great theoretical and practical significance for the treatment of PCH and the prevention and treatment of heart failure.
【學(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R542.2;R-332

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