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目的:探讨形成拟胚体(EBs)起始小鼠胚胎干细胞(ESCs)数量对心肌细胞及内皮细胞分化的影响。方法:将形成EBs起始ESCs数量为400,1 000,4 000的3个组(HD400,HD1000,HD4000)通过悬滴法促进其分化。免疫荧光检测TnT及CD31的表达;在不同时间点计算三个组搏动EBs百分比;RT-PCR检测Nkx2.5,GA-TA4,β-MHC,flk-1,CD31,tie-2基因表达水平。结果:通过悬滴法形成的EBs能够出现自发性搏动并且有TnT及CD31表达;HD1000组和HD4000组相比HD400组有更高的搏动EBs百分比及Nkx2.5,GATA4,β-MHC基因表达水平;HD400组相比HD1000组和HD4000组有更高的flk-1,CD31,tie-2基因表达水平(P<0.05)。结论:形成EBs起始ESCs数量是影响心肌细胞和内皮细胞分化的一个重要因素,起始细胞数量多则心肌细胞分化程度高,数量少则内皮细胞分化程度高。
Objective: To investigate the effect of embryonic stem cells (ESCs) derived from embryoid bodies (EBs) on the differentiation of cardiomyocytes and endothelial cells. Methods: Three groups (HD400, HD1000, HD4000) that formed the initial ESCs with EBs of 400, 1 000 and 4 000 were induced to proliferate by hanging drop method. The expression of TnT and CD31 were detected by immunofluorescence. The percentage of beating EBs in three groups was calculated at different time points. The expressions of Nkx2.5, GA-TA4, β-MHC, flk-1, CD31 and tie-2 were detected by RT- Results: The spontaneous beats and the expression of TnT and CD31 in EBs formed by the hanging drop method were higher than those in the HD4000 group. HD1000 group and HD400 group had higher percentage of beating EBs and Nkx2.5, GATA4 and β-MHC gene expression ; HD400 group had higher flk-1, CD31 and tie-2 gene expression levels (P <0.05) than HD1000 group and HD4000 group. CONCLUSION: The number of ESCs initiating EBs is an important factor that affects the differentiation of cardiomyocytes and endothelial cells. The number of initiating cells is higher than that of cardiomyocytes, and the number of ESCs is small.