BACKGROUND: Phosphoprteomic analyses of human embryonic stem cells (hESCs) in undifferentiated state and their differentiated derivatives have become a hotspot. The researchs suggest that phosphorylation events determine hESCs'fate. OBJECTIVE: To investigate the expression of Pten Mrna and the key proteins of PTEN/Akt/Mtor signaling in human embryonic stem cells in order to provide the basic research for stem cells expansion and differentiation. METHODS: The hESCs were cocultured with the murine fetal fibroblasts to maintain undifferentiation and digested by collagenase for detection. The growth morphology of hESCs mere observed. RT-PCR assay was used to determine Pten gene expression, and Western blot assay to detect the expression of p-PTEN, p-Mtor, p-P70S6K. P-4E-BP1 in hESCs. RESULTS AND CONCLUSION: The expression level of Pten was higher in hESCs and feeder cells than in K562. While the expression level of the key proteins in hESCs was lower than K562. Especially p-4E-BP1 in hESCs. Over-activation of the PI3K/Akt/Mtor signaling may accelerate the proliferation of hESCs and deduce the apoptosis, and provide more cells for differentiation and regeneration medicine.%背景:各种磷酸化蛋白质表达水平对人胚胎干细胞维持未分化状态或定向分化的影响逐渐成为人胚胎干细胞的研究热点,研究发现磷酸化蛋白质表达水平可能决定着人胚胎干细胞的命运.目的:对PI3K/Akt途径下游的关键蛋白磷酸化水平进行检测,寻找PI3K/Akt途径中能够维持人胚胎干细胞未分化状态的下游蛋白.方法:用胎鼠成纤维细胞作为饲养层,二维培养的方法培养人胚胎干细胞,胶原酶消化后待测;以饲养层细胞、K562细胞株为对照.观察人胚胎干细胞生长状态;RT-PCR检测Pten基因表达;Western Blot检测p-PTEN、p-mTOR、p-P70S6K、p-4E-BP1 4种蛋白的表达.结果与结论:人胚胎干细胞在未分化状态时Pten mRNA表达高于肿瘤细胞K562,而且Pten抑制的mTOR信号通路中关键蛋白表达均明显低于K562,尤其以p-4E-BP1表达最低.提示PI3K/Akt/mTOR信号通路下游磷酸化蛋白在人胚胎干细胞活性较低,如果抑制负调节蛋白PTEN或直接激活该通路正调节关键蛋白,可能会加快人胚胎干细胞增殖、减少凋亡,进而为定向分化、再生医学提供更多的细胞来源.
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