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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 isolate and identify neonatal rat cardiac progenitor cells (CPCs). Methods Primary culture of CPCs was performed by differential adherence. Morphological observation and cell proliferation were detected by CFDA-SE. The Fluo-3 / AM calcium ion detection method was used to detect the change of calcium concentration in colony proliferating cells. Immunofluorescence was used to detect the expression of stem cell markers and cardiomyocyte markers in colony proliferating cells. Results Differential adherence method was used to obtain the cells whose colonies proliferated about 1 week after cell culture. The round / oval or irregular polygonal cells were arranged in paving way. With the training time, cell colonies increased. After cultured for about 3 weeks, the morphology of the cells in the cell colonies changed (differentiated), and gradually emerged or spindle-shaped. The fluctuating / beating cardiomyocytes appeared in the colonies. CFDA SE tracing showed that the cells showed colony-proliferating characteristics. Fluo-3 / AM calcium detection showed that the concentration of calcium in differentiated cells in colony-proliferating cells increased. Immunofluorescence showed that the different colony proliferating cells were heterogeneous with the presence of c-kit ~ + / CD34 ~ -, Nanog ~ + / CD34 ~ - and c-kit ~ - / Nanog ~ Co-expression of c-kit and Gata4 was present, and cardiac troponin T (cTn T) cell colonies appeared as the cells differentiated. Conclusions Primary progenitor cells differentiated by differential adherence method have the ability to proliferate and differentiate into cardiomyocytes, which is the ideal material for studying the surface markers, proliferation and regulatory mechanisms of cardiac progenitor cells.