新生大鼠心祖细胞原代分离培养及鉴定

Primary culture and identification of cardiac progenitor cells of neonatal rats

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。

Objective To primarily culture and identify the cardiac progenitor cells（ CPCs） in vitro with morphological observation, immunofluorescence and tracing technology. Methods The CPCs were separated and cultivated with differential adhesion methods. The proliferation of CPCs was traced with two carboxyfluorescein succinimidyl ester（ CFDA-SE） tracer,observed with a phase contrast microscope. The calcium ion concentration of CPCs was detected with Fluo-3 / AM. The expressions of stem cell markers and cardiomyocyte markers were detected with immunoflurescent staining in colony proliferation cells. Results The CPCs of primary culture in vitro with differential adhesion methods showed colony proliferation, in which the undifferentiated cells appeared as circle, oval or irregular and took the arrangement of cobblestone appearance. The cell colony was bigger and the differentiated cells extended to protrude or showed spindles with the extension of incubation time. About 3 weeks,there were the beating myocardial cells in the colony proliferating cells. The CFDA-SE tracing showed that the fluorescence of colony proliferating cells decreased several times.Fluo-3 / AM calcium ion inspection showed that its concentration in cardiomyocyte-like cells of colony proliferating cells was more than that in undifferentiated CPCs. Immunoflurescence staining appeared as heterogeneous populations in colony proliferating cells,including c-kit~＋/ CD34^-,Nanog~＋/ CD34^-and c-kit~＋/ Gata4~＋colony. The colony proliferation cells showed cardiac troponin T（ c Tn T） positive expression. Conclusion The CPCs isolated in vitro with the differential adhesion methods have the characteristic of colony proliferation and differenciation, which can differentiate into cardiomyocytes. They are the best cells to research the biological characteristic,such as,the surface marker,the proliferation mechanism and regulation mechanism.