mTOR信号通路在iPS定向分化RPE细胞中的调控机制研究

The mechanism of mTOR signaling pathway in the regulation of differentiation of iPS into retinal pigment epithelial cells

目的探讨mTOR信号通路在iPS定向分化RPE细胞中的调控机制。方法培养iPS细胞,悬浮培养后形成拟胚体EB,诱导分化为RPE细胞。通过免疫细胞化学的方法,观察iPS-RPE细胞分化一个月后特异性蛋白(RPE65、LRAT、zo-1)的表达。同时通过Q-PCR,Western Blotting的方法检测iPS-RPE在不同分化时间点(分化1个月、2个月、3个月)上,iPS-RPE细胞中特异性基因、蛋白的表达变化以及mTOR信号通路的活性。最后应用雷帕霉素抑制iPS-RPE细胞中mTOR的通路,进一步观察iPS-RPE细胞中的蛋白表达变化。Q-PCR采用单因素方差分析(One-Way ANOVA)进行组间比较。结果荧光显微镜观察到iPS-RPE细胞在分化1个月时,表达RPE65、LRAT、zo-1蛋白。与对照组iPS比较,Q-PCR结果显示,实验组RPE65在分化1个月,2个月,3个月时的表达量分别为0.84±0.13,4.8±1.1,20.3±4.9(P=0.000);Best1分化1个月,2个月,3个月时的表达量分别为1.5±0.16,2.3±0.68,11.78±1.57(P=0.000);MerTK在分化1个月,2个月,3个月时的表达量分别为4.12±1.94,1.87±0.76,15.53±1.33(P=0.000),CK18分化1个月,2个月,3个月时的表达量分别为2.4±0.63,2.3±0.37,9.67±1.44(P=0.000),Western Blotting结果也表明,随着分化时间延长,iPS-RPE细胞中特异性蛋白(BEST1、catenin、MerTK)表达量有显著提高,而mTOR信号通路的活性受到抑制。与对照组(加DMSO)比较,雷帕霉素处理获得的iPS-RPE细胞中BEST1、MerTK、ck18蛋白表达量上升不是很明显,catenin蛋白显著提高。结论建立了体外高等分化、功能高效的iPS-RPE细胞,随iPS-RPE细胞分化时间延长,mTOR信号通路的活性是逐步抑制的。

Objective To explore the role of mTOR signaling pathway in the regulation of differentiation of iPS into retinal pigment epithelial （RPE） cells. Methods After embryoid bodies were formed by cultured iPS in suspension condition, they were induced to differentiate into RPE cells. The expressions of RPE specific proteins （RPE65, LRAT, ZO-1） in iPS-RPE cells during differentiation were detected by immunocytochemistry. Q-PCR and Western Blotting were carried out to analyze RPE specific genes, proteins and mTOR activity in iPS-RPE at different time points of differentiation （after one month, two months, three months）. Finally, iPS-RPE were treated with mTOR inhibitor rapamycin and RPE specific protein expression was evaluated. Results The RPE specific proteins （RPE65, LRAT, zo-1） of iPS-RPE cells were observed after one month of differentiation by fluorescence microscope. Compared with the control iPS, Q-PCR results showed that iPS-RPE exhibited significant higher level of RPE specific genes （RPE65, Best1, MerTK, CK18） after three months of differentiation （P〈0.01）. Western Blotting also showed that the expressions of RPE specific proteins BEST1, catenin and MerTK significantly increased in iPS-RPE cells as differentiation process went on. The activity of the mTOR was inhibited in this process. In addition, rapamycin treated cells exhibited higher expression of catenin, but expression of BEST1, MerTK and CK18 was not changed. Conclusions We established an efficient method to obtain iPS-RPE cells. And mTOR signaling pathway is gradually suppressed during the process of iPS differentiation into RPE cells in vitro.