新生大鼠心祖细胞原代分离培养及鉴定

发布时间：2018-03-10 20:23

本文选题：心祖细胞　切入点：集落增殖　出处：《解剖学报》2016年02期 　论文类型：期刊论文

【摘要】：目的原代分离培养及鉴定新生大鼠心祖细胞(CPCs)。方法利用差速贴壁原代分离培养CPCs,形态观察和二乙酸羧基荧光素-琥珀酰亚胺酯(CFDA-SE)示踪法检测细胞增殖。使用Fluo-3/AM钙离子检测法,检测集落增殖细胞中钙离子浓度变化。免疫荧光技术检测集落增殖细胞的干细胞标记物及心肌细胞标记物的表达。结果差速贴壁法获得细胞培养1周左右即观察到集落增殖的细胞,圆/椭圆形或不规则多边形细胞呈铺路石样排列。随培养时间延长,细胞集落增大。培养3周左右,细胞集落中细胞形态发生改变(分化),逐渐出现突起或呈梭形等,集落中出现波动/跳动的心肌细胞。用CFDA SE示踪显示,细胞呈集落增殖特点;Fluo-3/AM钙离子检测显示,集落增殖细胞中分化细胞的钙离子浓度增高。免疫荧光检测显示,不同的集落增殖细胞具异质性,存在c-kit~+/CD34~-、Nanog~+/CD34~-和c-kit~-/Nanog~-细胞集落;增殖的细胞集落存在c-kit和Gata4阳性共表达,随细胞分化出现心肌肌钙蛋白T(c Tn T)细胞集落。结论差速贴壁法原代分离培养的心祖细胞具有集落增殖和分化为心肌细胞的能力,是研究心祖细胞表面标记物、增殖和调控机制的理想材料。
[Abstract]:Objective to isolate and identify neonatal rat cardiac progenitor cells (CPCs). Methods Fluo-3/AM calcium ion assay was used to detect the proliferation of neonatal rat cardiac progenitor cells. The changes of calcium concentration in colony proliferating cells and the expression of stem cell markers and myocardial cell markers in colony proliferating cells were detected by immunofluorescence technique. To colony proliferating cells, Round / oval or irregular polygon cells were arranged like paving stones. With the extension of culture time, the cell colony increased. After 3 weeks of culture, the cell morphology of the cell colony changed (differentiation and gradually appeared protuberance or fusiform, etc.). There were fluctuating / beating cardiomyocytes in the colony. CFDA SE tracing showed that the colony proliferation was the characteristic of Fluo-3 / AM Ca ~ (2 +), and the calcium concentration of differentiated cells in colony proliferating cells was increased. Different colony proliferative cells have heterogeneity, there are c-kit~ / / CD34 ~ / CD34 ~ + and c-kit-r / Nanog-cell colonies, and c-kit and Gata4 positive coexpression exists in the proliferative colonies, and the positive expression of c-kit and Gata4 is also found in the proliferative cell colonies, and the positive expression of c-kit and Gata4 is also found in the proliferative colonies. Conclusion Cardiac progenitor cells isolated and cultured by differential adherent method have the ability of colony proliferation and differentiation into cardiomyocytes, which are the surface markers of cardiac progenitor cells. Ideal material for proliferation and regulatory mechanisms.
【作者单位】：河南省医用组织再生重点实验室;新乡医学院第一附属医院心内科;
【基金】：新乡医学院科研项目培育基金(2013ZD121) 2012年度研究生科研创新支持计划
【分类号】：R329.2

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