扩张型心肌病诱导多潜能干细胞模型的建立

发布时间：2019-02-17 17:08

【摘要】：扩张型心肌病(Dilated Cardiomyopathy, DCM)是一类由原发性心肌病变导致的心肌疾病,临床特征为左心室、右心室或双侧心室扩张,并伴有心肌肥厚、心室收缩功能障碍,伴或不伴充血性心力衰竭等。其病情呈进行性加重,死亡可发生于疾病的任何阶段,它是导致心力衰竭的主要原因之一。DCM的发病机制目前尚不清楚,病毒感染、自身免疫、细胞凋亡、遗传等因素可能与DCM的发病相关。部分DCM有明显的家族聚集性,通过家系调查和超声心动图对DCM患者的亲属筛查证实,约25%-35%的DCM为家族性扩张型心肌病(familial dilated cardiomyopathy, FDCM)。已经观察到FDCM有常染色体显性遗传、常染色体隐性遗传、X-连锁遗传、母系遗传等遗传方式。FDCM为DCM遗传基础及致病机理的研究提供了独特的资源。但是,由于很难获得病变组织标本,无法开展相关致病机理研究,这在很大程度上制约了DCM发病机制的研究。 2006年日本科学家Shinya Yamanaka首先报道通过外源表达Oct4,Sox2,Klf4和c-Myc四个因子可以将小鼠胚胎成纤维细胞(MEF)转化成诱导多潜能干细胞(iPSCs),2007年Yamanaka实验室和Thomson实验室独立报道了在人类细胞中诱导多潜能干细胞获得成功。iPSCs在形态、全基因组表达谱、表观遗传学以及分化潜能等方面与胚胎干细胞(ESCs)十分相似。iPSCs取材方便,并且具有与ESCs相同的分化潜能,能够分化成有功能的心肌细胞,可为DCM的病理机制研究带来新的策略。本实验室前期研究工作中收集到一个伴发有传导功能障碍的扩张型心肌病家系,呈常染色体显性遗传。遗传学研究发现TNIKc.153AG的杂合突变在家系中与疾病共分离,为了在心肌细胞中研究该突变的致病机理,我们通过皮肤组织活检获得了患者的皮肤组织,经原代培养得到皮肤成纤维细胞(HDF),同时用293T细胞包装含Oct4,Sox2,Klf4和c-Myc四个因子的假病毒,通过病毒高效的将Oct4,Sox2,Klf4和c-Myc导入HDF中表达,同时改变培养条件、加入提高诱导效率的小分子化合物维生素C和丙戊酸,进行iPSCs的诱导,获得了ESCs样的克隆。我们挑取的克隆能够用ESCs培养条件扩增,始终保持ESCs样的形态特征,通过AP染色、核型鉴定、突变验证、免疫荧光检测、多潜能性相关基因表达分析和体内体外分化能力检测确定了我们获得的细胞为来自HDF的完全重编程的iPSCs,建立了该FDCM的诱导多潜能干细胞模型。同时用ESCs摸索多能干细胞向心肌分化的方法,以建立高效的iPSCs向心肌分化的方法,但是我们的心肌分化只在ESCs中取得了成功,后续研究工作将致力于iPSCs向心肌细胞的分化。
[Abstract]:Dilated cardiomyopathy (Dilated Cardiomyopathy, DCM) is a kind of myocardial disease caused by primary cardiomyopathy. It is characterized by dilatation of left ventricle, right ventricle or bilateral ventricle, accompanied by hypertrophy of myocardium and dysfunction of ventricular systolic function. With or without congestive heart failure, etc. Its condition is progressive aggravation, death can occur at any stage of the disease, it is one of the main causes of heart failure. The pathogenesis of DCM is not clear, virus infection, autoimmune, apoptosis, Genetic factors may be associated with the pathogenesis of DCM. Some DCM had obvious familial aggregation. Family investigation and echocardiographic screening of relatives of DCM patients confirmed that about 25-35% of DCM was familial dilated cardiomyopathy (familial dilated cardiomyopathy, FDCM). Autosomal dominant inheritance, autosomal recessive inheritance, X-linkage inheritance and maternal inheritance have been observed in FDCM. FDCM provides a unique resource for the study of genetic basis and pathogenesis of DCM. However, because it is difficult to obtain pathological tissue samples, it is impossible to study the pathogenesis of DCM, which restricts the study of pathogenesis of DCM to a great extent. In 2006, Japanese scientist Shinya Yamanaka first reported that the expression of four factors, Oct4,Sox2,Klf4 and c-Myc, could transform mouse embryonic fibroblasts (MEF) into induced multipotent stem cells (iPSCs),). In 2007, Yamanaka Labs and Thomson Labs independently reported the success of inducing pluripotent stem cells in human cells. Epigenetics and differentiation potential are very similar to embryonic stem cells (ESCs). IPSCs has the same differentiation potential as ESCs and can differentiate into functional cardiomyocytes. It can bring new strategies for the study of pathological mechanism of DCM. An autosomal dominant inheritance of dilated cardiomyopathy (DCM) with conduction dysfunction was collected in our laboratory. Genetic studies have found that the heterozygous mutation of TNIKc.153AG is isolated from the disease at home. In order to study the pathogenesis of the mutation in cardiomyocytes, we obtained the skin tissue of the patient by skin biopsy. Skin fibroblasts (HDF),) were obtained by primary culture, and 293T cells were used to package pseudoviruses containing Oct4,Sox2,Klf4 and c-Myc. Oct4,Sox2,Klf4 and c-Myc were highly expressed in HDF. At the same time, the iPSCs was induced by adding vitamin C and valproic acid, which could improve the induction efficiency. The ESCs like clones were obtained. The clones we selected can be amplified by ESCs culture conditions, always maintaining the morphological characteristics of ESCs, by AP staining, karyotype identification, mutation verification, immunofluorescence detection, The multipotential gene expression analysis and in vitro differentiation test confirmed that the cells we obtained established the FDCM induced multipotential stem cell model for the fully reprogrammed iPSCs, from HDF. At the same time, ESCs was used to explore the method of multipotent stem cell differentiation into myocardium, so as to establish an efficient method for the differentiation of iPSCs into myocardium. However, our myocardial differentiation was only successful in ESCs. The subsequent research will focus on the differentiation of iPSCs into cardiomyocytes.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R542.2;R-332

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