拟胚体贴壁时间对小鼠胚胎干细胞心肌分化的影响

Effect of embryoid body adhesion time on the cardiac differentiation of mouse embryonic stem cells

目的观察拟胚体(EB)贴壁时间对小鼠胚胎干细胞(ESC)心肌分化的影响并研究其机制。方法用悬滴培养法促进ESCs形成EBs。EBs在不同的分化天数贴壁,观察搏动EBs百分比,RT-PCR检测Nkx2.5,GATA4和β-MHC mRNA表达,Western blot检测Src家族酪氨酸激酶磷酸化水平。结果分化第3,4天贴壁组搏动EB百分比及Nkx2.5,GATA4,β-MHC表达水平显著低于分化第5,6和7天贴壁组(P<0.05);EB贴壁能够使Src激酶磷酸化水平升高,Src激酶阻断剂PP2能够抑制贴壁诱发的Src激酶磷酸化水平升高(P<0.05);对于分化第4天贴壁组,在分化第4～6天使用PP2能够增加搏动EBs百分比及β-MHC表达水平(P<0.05)。结论 EB贴壁时间是影响ESC心肌分化的一个重要因素。在分化第4天或者之前贴壁可以显著抑制心肌分化,其机制可能是EB贴壁激活了Src激酶。

Objective To investigate the time-dependent effect of embryoid body （EB） adhesion on the cardiac differentiation of mouse embryonic stem cells （ESCs） and its mechanisms. Methods Hanging drop culture was used to induce ESCs differentiating into cardiomyocytes through forming EBs. Suspended EBs were transferred onto tissue culture plates at different differentiation days. The percentage of beating EBs was calculated at different time points. The mRNA expression of Nkx2. 5, GATA4 and β-MHC were detected by RT-PCR. The phospho- rylation of Src kinase was detected by Western-blot. Results The groups with EB adhesion times of 3 or 4 days had higher percentage of beating EBs and higher gene expression of Nkx2. 5, GATA4 and β-MHC compared with those groups with EB adhesion times of day 5, 6 or 7 （ P 〈 0.05 ）. EB adhesion triggered the Src kinase, which was inhibited by PP2 （P 〈 0. 05 ）. Furthermore, the percentage of beating phosphorylation of EBs and gene expression of fl-MHC were enhanced by PP2 at differentiation day 4 - 6 in the group whose EB adhesion time was at differentiation day 4 （ P 〈 0.05 ）. Conclusions The EB adhesion time is important in regulation of the cardiac differentiation of ESCs. When EBs adhere to gelatin-coated plates at differentiation day 4 or earlier, the cardiac differentiation is significantly inhibited. The mechanism is likely that EB adhesion triggers the phosphorylation of Src kinase.