神经调节蛋白1诱导小鼠胚胎干细胞向心肌细胞的分化及其机制探讨

摘要

背景:前期研究发现神经调节蛋白1早期干预能够诱导胚胎干细胞向心肌细胞分化.目的:进一步观察神经调节蛋白1诱导小鼠胚胎干细胞向心肌细胞分化的途径.方法:分别观察胚胎干细胞自发与在神经调节蛋白1诱导情况下向心肌细胞的分化,RT-PCR检测胚胎干细胞自发与诱导分化下心肌特异性早期转录因子GATA-4、Nkx2.5 mRNA的表达,细胞免疫组织化学检测胚胎干细胞自发与诱导分化下心肌肌钙蛋白T及磷酸化蛋白激酶B的表达,Western blot半定量分析自发与诱导分化下心肌细胞磷酸化蛋白激酶B的表达.结果与结论:神经调节蛋白1诱导心肌分化率显著高于自发心肌分化率.神经调节蛋白1呈剂量依赖性上调GATA-4、Nkx2.5 mRNA表达;表皮生长因子样受体拮抗剂及磷脂酰肌醇-3激酶抑制剂阻断了神经调节蛋白1对Nkx2.5 mRNA的表达上调作用.Western blot分析显示神经调节蛋白1诱导组拟胚体中自发性搏动心肌细胞团磷酸化蛋白激酶B的相对表达高于自发心肌分化组.提示神经调节蛋白1可能通过磷脂酰肌醇3激酶/磷酸化蛋白激酶B信号通路诱导胚胎干细胞向心肌细胞分化.%BACKGROUND: Previous studies demonstrated that the early invention of neuregulin-1 (NRG-1) can induce embryonic stem cells (ESCs) to differentiate into cardiomyocytes. OBJECTIVE: To further investigate the pathway of NRG-1 induces embryonic stem cells to differentiate into cardiomyocytes. METHODS: The process of cardiomyocytes (CMs) differentiated from ESCs with or without NRG-1 treatment was observed continuously. RT-PCR was performed to detect the mRNA expression of early cardiac-restricted transcript factors Nkx2.5 and GATA-4 during differentiation. The protein expression of cardiac troponin T and phosphorylated protein kinase B was determined by immunhistochemistry (IHC), phosphorylated protein kinase B expression of CMs was analyzed by semiquantitative western blot analysis. RESULTS AND CONCLUSION: The differentiation rate of CMs induced by NRG-1 was significantly higher than spontaneous CMs differentiation rate. NRG-1 exhibited a dose dependent up regulatory effect on the expression of GATA-4, Nkx2.5 mRNA, while NRG-1-induced increase of Nkx2.5 transcripts was inhibited by treatment with receptor antagonist and phosphatidylinositol 3-kinase (PI3K) inhibitor. Western blot analysis confirmed that the expression of phosphorylated protein kinase B of spontaneous beating CMs in embryoid body in NRG-1 induced group was relatively higher than that in spontaneous CMs differentiation group. The results suggested that NRG-1 promotes CMs differentiation of mouse ESCs via PI3K/phosphorylated protein kinase B/ phosphorylated protein kinase B activation.