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目的研究青-链霉素(双抗)对小鼠胚胎干细胞(mouse embryonic stem cells,mESCs)向心肌细胞分化的影响。方法采用常规悬滴法培养mESC形成拟胚体(embryonic body,EB),在分化培养基中加入不同浓度的双抗(1.0%、5.0%和10.0%)培养贴壁的EB,选择不同的时间点分化的细胞通过流式细胞术(FCM)、细胞免疫荧光、westem blotting等分析心肌特异性蛋白表达水平的差异。结果小鼠多能干细胞表面标志物SSEA-1表达呈阳性(绿色),H.E染色显示核质比>>1。正常自分化细胞免疫荧光显示cTnT、‘MLC2阳性(绿色),肌节清晰可见。高浓度双抗组,cTnT+阳性细胞率高于对照组和低浓度(1.0%)双抗组,western blotting显示其心肌特异性蛋白cTnT、cTnI、MLC2、connexin43等表达增加(p<0.05)。结论高浓度双抗促进mESCs向心肌细胞分化,且有浓度依赖性。
Objective To investigate the effect of cyanamide (streptomycin) on the differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes. Methods MESCs were cultured by conventional hanging drop method to form embryonic body (EB) .Adding different concentrations of double antibody (1.0%, 5.0% and 10.0%) to adherent EBs in differentiation medium, different time Differentiated cells were analyzed for differences in myocardial specific protein expression by flow cytometry (FCM), immunofluorescence, and western blotting. Results The expression of SSEA-1 on mouse pluripotent stem cells was positive (green), and H.E staining showed that the nuclear mass was> 1. Normal autodifferentiated cell immunofluorescence showed cTnT, ’MLC2 positive (green), sarcomere clearly visible. The positive rate of cTnT + cells in high concentration double antibody group was higher than that in control group and low concentration (1.0%) double antibody group. The expression of cTnT, cTnI, MLC2 and connexin43 were increased by Western blotting (p <0.05). Conclusion High concentration double antibody can promote the differentiation of mESCs into cardiomyocytes in a concentration-dependent manner.