人类胚胎干细胞来源的心肌细胞模型的建立及系统鉴定方案

发布时间：2018-11-28 08:50

【摘要】：心肌细胞是研究心血管疾病的重要工具之一,但是人类心肌细胞较难获得和培养.为人类胚胎干细胞诱导分化成心肌细胞提供一个有用的实验方法和鉴定方案.人胚胎干细胞以其多向分化的特性为体外研究提供了细胞资源.在人胚胎干细胞诱导分化为心肌细胞的过程中,通过荧光定量聚合酶链式反应(polymerase chain reaction,PCR)检测发现,在分化过程中干细胞标记物Cripto,Dnmt3b,Wnt3,KIF4,Oct4,SOX2和Nanog表达下降,心肌特异性结构蛋白cTnT和α-actinin以及心脏前体细胞分化标记物Nkx2.5表达上升.分化完成后用免疫荧光检测心肌特异性结构蛋白Tnn T2和α-actinin,通过分析Tnn T2阳性细胞的比例,α-actinin阳性细胞的比例,以及Tnn T2和α-actinin双阳性细胞的比例发现,在所提出的胚胎干细胞诱导分化体系中,三者比例分别为90.80%,91.00%,90.91%,表明在此诱导分化条件下人胚胎干细胞可成功分化成为心肌细胞.成功建立了人胚胎干细胞来源的心肌细胞模型以及基于标记物荧光定量PCR及免疫荧光系统检测的鉴定方案,为未来心血管疾病的基础研究及心脏毒性药物检测奠定了一定的基础.
[Abstract]:Cardiomyocytes are one of the important tools to study cardiovascular disease, but human cardiomyocytes are difficult to obtain and culture. To provide a useful experimental method and identification scheme for human embryonic stem cells to differentiate into cardiomyocytes. Human embryonic stem cells provide cell resources for the study in vitro because of their multidirectional differentiation. In the process of inducing human embryonic stem cells to differentiate into cardiomyocytes, the expression of Cripto,Dnmt3b,Wnt3,KIF4,Oct4,SOX2 and Nanog, the markers of stem cells, decreased in the process of differentiation by fluorescence quantitative polymerase chain reaction (polymerase chain reaction,PCR). The expressions of myocardial specific structural proteins (cTnT and 伪-actinin) and cardiac precursor cell differentiation marker (Nkx2.5) were increased. After differentiation, the ratio of Tnn T2 positive cells, 伪 -actinin positive cells and Tnn T2 and 伪 -actinin double positive cells were detected by immunofluorescence. In the system of inducing differentiation of embryonic stem cells, the ratio of the three was 90.80,91.00 and 90.91respectively, which indicated that human embryonic stem cells could be successfully differentiated into cardiomyocytes under the condition of inducing differentiation. The cardiac myocyte model derived from human embryonic stem cells and the identification scheme based on fluorescent quantitative PCR and immunofluorescence system were successfully established. It will lay a foundation for the future basic research of cardiovascular disease and the detection of cardiac toxic drugs.
【作者单位】：南京医科大学第一附属医院心脏内科;
【基金】：国家自然科学基金资助项目(81370280,81570332)
【分类号】：R54

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