利用小鼠过表达模型研究hHole和Pygo1基因在病理性心肌肥厚发生中的作用

发布时间：2018-06-25 10:02

本文选题：hHole + Pygopus1　；参考：《湖南师范大学》2016年博士论文

【摘要】：病理性心肌肥厚(Pathological Cardiac Hypertrophy,PCH)是临床各类心脏疾病发展终末期的一个共同病理过程。据报道,该疾病在全球范围内的人群发病率约0.2%,在我国的成人群体中发病率约0.18%,约100万患者,且呈逐年增长趋势。PCH的发生、发展涉及多基因、多通路、多环节及多层次,迄今至少已发现有20个基因1400多种突变与PCH的发生相关。但仍有许多调控基因有待鉴定,其发生、发展的分子机制亦有待阐明。hHole与Pygo1均是在脊椎动物成体心脏组织中特异性高表达的基因,我们的前期研究结果显示在人类心衰的心肌标本中,hHole和Pygo1的均出现异常表达,然而对于hHole和Pygo1基因功能的研究,尤其是在心脏中的功能研究,迄今国际上尚未见相关报道。因此本文首先应用生物信息学方法,对人HOLE及人PYGO1蛋白的基本理化性质、结构特征等进行预测分析,并进一步通过体内遗传修饰手段构建心肌特异性过表达的转基因动物模型,旨在通过建立hHole和Pygo1过表达转基因小鼠模型,分别探讨hHole和Pygo1在PCH发生中的作用,并进一步阐明其潜在的作用机理。一、心肌特异性过表达hHole阻遏ISO诱导的病理性心肌肥厚Hole基因作为一个在心脏发育和成体期均有高表达的基因,已经被发现十多年,但国际同行们似乎一直忽视了它在体内的生物学功能,Hole基因的生物学功能迄今仍是一片空白。我们的前期研究结果揭示,HOLE蛋白具有ERK结合位点(D-domain)和结合SH3结构域的多聚脯氨酸序列(PXXP),且上述结构域对SRF肥大信号具有一定的抑制作用。此外,有报道发现人类hHole基因(TMEM121)内部PXXP位点的SNP与人类先天性心脏病存在一定的相关性,似乎使得hHole基因在心脏中的功能变得可以预知。在前期研究中,我们还发现在人类扩张型心肌病患者中,hHole mRNA表达显著升高,进一步在体内外水平分析心肌肥厚的小鼠模型及心肌细胞肥大的细胞模型中Hole的表达水平,发现Hole在mRNA和蛋白水平较对照组均有显著升高,上述研究结果提示Hole可能参与病理性心肌肥厚的调控。为研究hHole基因在病理性心肌肥厚的发生、发展过程中的作用,我们首先应用生物信息学方法对人类hHole基因的基本结构、理化性质等进行了预测分析,其次,为从实验水平验证人类hHole基因的相关功能,我们应用体内遗传修饰手段,首次建立了心脏特异性hHole过表达转基因小鼠模型。我们发现转基因小鼠在正常的生理条件下,表型正常,但效应于异丙肾上腺(ISO)刺激,转基因的小鼠较WT小鼠表现出抵抗ISO诱发的心肌肥厚,具体表现为心肌细胞面积的细微变化、轻微的心肌纤维沉积、良好的心功能输出等。上述结果初步证实hHole基因可能是PCH的抑制因子,我们的研究结果首次从分子水平上揭示hHole基因在脊椎动物成体内的生物学功能。为深入研究hHole抑制病理性心肌肥厚的作用机理,我们进一步应用q RT-PCR方法分析TG小鼠中MAPK家族成员在mRNA水平的表达变化,结果发现ISO刺激的转基因小鼠中ERK1,ERK2的mRNA表达水平均有下调,JNK或P38在mRNA水平则无显著性变化。进一步应用W B方法检测转基因小鼠心脏组织中p-ERK及t-ERK的表达变化,结果显示:效应于ISO刺激,心肌特异性过表达hHole可显著抑制ERK1/2的磷酸化。上述研究结果提示hHole可能通过ERKs信号途经在转录和翻译后水平双重调控病理性心肌肥厚。二、心肌特异性过表达Pygo1自主调控病理性心肌肥厚果蝇Pygopus基因是2002年发现的经典WNT信号通路的关键成员。具有调控组织发育、染色质重塑、转录激活等多种重要生物学功能。在脊椎动物中Pygopus有两种同源基因,分别是Pygopus1和Pygopus2(Pygo1,Pygo2)。在成体小鼠中,Pygo2基因在多种组织中广泛表达,但Pygo1仅在心脏组织中特异性高表达。目前对Pygo1的研究相对甚少,其在脊椎动物成体心脏中的功能研究更是一片空白。Pygo1基因在成体心脏中特异性高表达是否提示其可能与心脏功能密切相关?前期研究发现人类心肌肥厚患者中PGYO1蛋白表达异常升高,心肌肥厚小鼠模型中Pygo1在mRNA和蛋白表达与“胚胎基因”ANF、β-MHC、SK-α-actin的表达呈一致性上调。进一步在体外细胞水平的研究结果发现稳定过表达Pygo1的细胞系无需其它病理性肥厚刺激因子,即表现出细胞体积的增大和肥厚“标志基因”的上调;而在干扰Pygo1表达的细胞系中,被干扰细胞系可抑制由ISO或胎牛血清诱导的心肌肥大。以上结果提示Pygo1基因可能是调控PCH发生、发展的一个关键性作用因子。为深入研究Pygo1的生物学功能,我们首先应用生物信息学方法对人类Pygo1基因的基本结构及理化性质进行预测分析。其次,为进一步证实该基因在动物整体水平的表达效应,我们将Pgyo1基因构建在α-MHC启动子之下,并通过囊胚注射获得了过表达Pygo1的转基因小鼠后代,初步研究发现不需要任何肥大病理因子的刺激,Pygo1过表达成年小鼠中的“胚胎基因”ANP、β-MHC、SK-α-actin在mRNA水平的表达显著性上调,并伴有心肌细胞体积的增大及年龄相关的心肌纤维化等。我们的研究结果首次揭示Pygo1基因可自主调控PCH的发生,但其作用机理有待进一步的研究。病理性心肌肥厚的发生、发展机制非常复杂,我们首次应用生物信息学方法预测分析人hHole及Pygo1基因的基本理化性质及基本结构等,并在动物整体水平验证了Hole及Pygo1在小鼠成体心脏内的生物学功能。发现hHole可通过ERK信号途径在转录和翻译后水平双重抑制PCH的发生、发展。而心肌特异性过表达Pygo1可自主诱导PCH的发生。随着研究的深入,PCH的靶点干预似有曙光。后续研究中进一步从分子水平研究其关键靶点及开发靶向性药物,对于PCH的治疗及心力衰竭的防治将具重要的理论和现实意义。
[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.
【学位授予单位】：湖南师范大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R542.2;R-332

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