维生素C联合内皮素－1促进胚胎干细胞向心肌细胞分化及机制

摘要

目的：探讨维生素（Vit）－C联合内皮素（ET）－1对胚胎干细胞（ESC）向心肌细胞分化的影响及其可能机制。方法体外培养 ESC并给予Vit－C，摸索Vit－C作用于ESC的最佳浓度和干预时间；然后给予ET－1（100 nmol／L）或Vit－C（100μmol／L）＋ET－1（100 nmol／L），观察两组对拟胚体搏动、Nkx 2．5、GATA－4及Shox2表达的影响。同时，以RNA干扰方法沉默Nkx 2．5、GATA－4及 Shox2表达，观察沉默相应启动子表达水平后对Vit－C联合ET－1介导的拟胚体搏动增加的影响。结果 Vit－C最佳作用浓度为100μmol／L，最佳干预时间为分化的第4～9天，可使拟胚体搏动增加11．19％，同时上调拟胚体中心肌细胞标志物cTnT以及心肌发育启动子Nkx 2．5、GATA－4及Shox2表达。在Vit－C基础上进一步给予ET－1，可进一步使拟胚体搏动增加11．07％，同时进一步上调 cTnT、Nkx 2．5、GATA－4及 Shox2表达水平。沉默 Shox2基因表达使拟胚体搏动幅度减少最明显，达12．94％。结论 Vit－C联合ET－1可获得进一步纯化的心肌细胞，上调Shox2是其促进ESC分化的最重要机制。%Objective To explore the effects of vitamin ( Vit) -C in combination with endothelin ( ET) -1 on embryonic stem cell ( ESC) differentiation into cardiomyocytes and underline mechanism.Methods Different Vit-C concentrations and intervention time were tested to optimize Vit-C treatment.After that,Vit-C (100 μmol/L) alone or together with ET-1 (100 nmol/L) were administered to ESC at day 4~9 after differentiation.The beating of embryoid bodies ( EBs) were calculated and compared between groups.Expressions of cardiac marker,cTnT,cardiac promoter,Nkx 2.5,GATA-4 and Shox2 were determined.Lentivirus mediated RNA interference was used to knockdown expressions of Nkx 2.5,GATA-4 and Shox2 and EBs beating under Vit-C and ET-1 treatment were observed.Results Optimal Vit-C treating concentration was 100μmol/L and day 4~9 after differentiation was the best time to intervent,which increased EBs beating by 11.19%and increased expression of cTnT,Nkx 2.5,GATA-4 and Shox2 expressions at the same time.In combination with ET-1 further increased EBs beating by 11.07%and expression profiles of the above proteins.Knocking down of Shox2 reversed the effects of Vit-C and ET-1 at the grea-test extent,reduced EBs beating by 12.94%.Conclusions Vit-C plus ET-1 produce more purified cardiomyocytes from ESC differentiation. The up-regulation of Shox2 is the most important underline mechanism.