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目的探讨肝细胞生长因子(HGF)和类胰岛素样生长因子(IGF1)在外能否诱导心肌干细胞(CSCs)发生增殖并向心肌细胞定向分化。方法组织贴块法分离培养心肌干细胞,免疫荧光技术鉴定c-kit和CD34表达,流式细胞仪分选纯化c-kit+细胞,CFDA SE荧光探针示踪培养检测细胞增殖特征。实验分为单纯心肌干细胞组和与心肌共培养组,分别用HGF和IGF1干预,倒置显微镜观察细胞不同时期数量与形态的变化,活细胞工作站观察CFDA SE示踪剂荧光强弱,采集图像,进行统计学分析。免疫荧光技术检测Nkx2.5、心肌肌钙蛋白T(cTnT)的表达。结果单纯心肌干细胞组,生长因子刺激后心肌干细胞数量与形态均无明显变化。与心肌共培养组,细胞均发生增殖与形态变化,Nkx2.5、cTnT阳性表达,有个别心肌干细胞分化为自发搏动的心肌细胞。结论心肌干细胞与心肌细胞共培养条件下,HGF和IGF1能够促进心肌干细胞增殖,联合作用能够诱导心肌干细胞向心肌细胞分化。
Objective To investigate whether hepatocyte growth factor (HGF) and insulin-like growth factor (IGF1) can induce cardiac stem cells (CSCs) to proliferate and differentiate into cardiomyocytes. Methods Cardiac stem cells were isolated and cultured by tissue patch method. The expression of c-kit and CD34 was identified by immunofluorescence staining. The c-kit + cells were sorted by flow cytometry. The cell proliferation characteristics were detected by CFDA SE fluorescent probe. The experiment was divided into the group of simple cardiac stem cells and the group of co-cultured with cardiac muscle. HGF and IGF1 were intervened respectively. The numbers and morphological changes of cells were observed under inverted microscope. The fluorescence intensity of CFDA SE tracer was observed by living cell workstation. Statistical analysis. Immunofluorescence was used to detect the expression of Nkx2.5 and cardiac troponin T (cTnT). Results There were no significant changes in the number and morphology of cardiac stem cells in the simple cardiac stem cell group and the growth factor-stimulated group. Co-culture group with myocardium, proliferation and morphological changes occurred in cells, Nkx2.5, cTnT positive expression, a few cardiac stem cells into spontaneous beating myocardial cells. Conclusions Under the conditions of co-culture of cardiac stem cells and cardiomyocytes, HGF and IGF1 can promote the proliferation of cardiac stem cells. The combined effect can induce the differentiation of cardiac stem cells into cardiomyocytes.