形成拟胚体细胞数量对小鼠胚胎干细胞心肌细胞及内皮细胞分化的影响

Effect of the Starting Number of Cells in Embryoid Bodies on Cardiac and Endothelial Differentiation of Murine Embryonic Stem Cells

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 the starting number of murine embryonic stem cells （ESCs） in embryoid bodies （EBs） on the cardiac and endothelial differentiation. Methods： Hang- ing drop culture was used to induce ESCs differentiating into cardiomyocytes and endothelial cells. The groups of the EBs formed from 400, 1 000, and 4 000 ESCs were referred to as HD400, HD1000 and HD4000, respectively. TnT and CD31 were detected by immunocytochem- istry. The percentage of beating EBs was calculated at different time points. The mRNA expres- sion of cardiac specific gene Nkx2.5, GATA4, β-MHC and endothelial specific gene flk-1, CD31, and tie-2 were detected by RT-PCR. Results. Spontaneously beating EBs were positively stained with TnT or CD31. The percentage of beating EBs and Nkx2.5, GATA4,β-MHC gene expres- sion were higher in the HD4000 group and HD1000 group compared with HD400 group. Moreo- ver, the HD400 group had higher gene expression of flk-1, CD31, tie-2. Conclusion. Our resultsindicate that the starting number of ESCs in EBs is important in regulation of the cardiac and endothe- lial differentiation of ESCs. The cardiac differentiation is increased in the EBs by a larger number of ESCs, while the endothelial differentiation is increased in the EBs by a smaller number of ESCs.