环孢霉素A对小鼠胚胎干细胞向心肌细胞分化的影响及调控机制

Influence of Cyclosporine A on the differentiation of mouse embryonic stem cells into cardiomyocytes and its regulatory mechanism

目的探讨环孢霉素A(CSA)对小鼠胚胎干细胞(ESCs)向心肌细胞分化的影响及调控机制。方法应用分阶段胚胎干细胞向心肌细胞的分化系统,设ESCs自发组和CSA诱导组,用细胞免疫组织化学和流式细胞仪(FACS)评估心肌细胞分化率以及计数。细胞免疫化学检测CSA诱导出的心肌细胞标志物的表达。在ES细胞分化的不同阶段添加CSA,观察CSA是否特异地作用于某一特定的分化阶段。分别添加另外一种免疫抑制剂也是钙调磷酸酶抑制剂他克莫司(FK506)和T细胞核因子(NFAT)抑制剂11R-VIVIT,与添加CSA比较,比较各组的心肌细胞分化率。结果 ESCs自发组的心肌细胞分化率为(6.00±0.05)%,CSA诱导组为(60.00±0.08)%,两组比较,差异有统计学意义(P<0.05),且诱导生成的心肌细胞具备心肌细胞特有的特征。CSA特异性地作用于中胚层干细胞,促进其分化成心肌干细胞;CSA强力促进中胚层干细胞分化成心肌细胞增多,相反分化成的上皮细胞减少;添加FK506和11R-VIVIT后没有出现类似CSA的促心肌分化作用。结论 CSA特异地作用于中胚层干细胞,通过非NFAT径路,能够强力诱导ESCs向心肌细胞分化。

Objective To explore the influence of Cyclosporine-A（CSA） on the differentiation of mouse embryonic stem cells（ESCs） into cardiomyocytes and its mechanism of regulation.Methods The differentiation system of mouse embryonic stem cells into cardiomyocytes by stages was used,and ESCs spontaneous group and CSA induction group were designed.The differentiation rate and count of cardiomyocytes were evaluated by cell immunohistochemistry and flow cytometry（FACS）.The expression of CSA-induced cardiomyocyte markers was detected by cell immunohistochemistry.CSA was added at different stages of ES cell differentiation to observe whether CSA specifically acted on a particular differentiation phase.Another immunosuppressive agent including tacrolimus（calcineurin inhibitor：FK506） or 11R-VIVIT（nuclear factor of activated T cells：NFAT） was separately added in order to compare the differentiation rate of cardiomyocytes among the groups.Results In the ESCs spontaneous group,the differentiation rate of cardiomyocytes was（6.00±0.05）%,and the rate in the CSA-induction group was（60.00±0.08）%,which was displayed a statistical difference（P〈0.05）.The cardiomyocytes generated from induction possessed the unique characteristics.CSA specifically acted on mesodermal stem cells and promoted the differentiation of these cells into cardiac stem cells.The amount of myocardial cells was increased differentiated from mesodermal stem cells strongly promoted by CSA,while the number into epithelial cells was in decrease.After adding FK506 and 11R-VIVIT,there was no similar effect of CSA on promotion of cardiomyocyte differentiation.Conclusion Cyclosporine-A specifically acts on mesodermal stem cells and can powerfully induce these ESCs into the differentiated cardiomyocytes via non-NFAT pathway.