Wnt/β-catenin /TCF4信号通路在胚胎干细胞微环境诱导人角膜上皮细胞去分化中的调控机制

It is clear that the microenvironment or niche plays an important role in determining the fate of stem cells: being stem cells or differentiated. The adult cells can be dedifferentiated and retain the undifferentiated phenotype and high proliferative capacity via embryonic stem cell microenvironment. However, the mechanisms especially the intrinsic pathways controlling the fate of the cells in the different microenvironment are largely unknown. Wnt signaling pathway is critical to the growth, development and differentiation of the cells. We observed that embryonic stem cell microenvironment culture system induced human corneal epithelial cells dedifferentiated and found Wnt/integrinβ1- FAK- Akt signaling pathway played an important role in this process. Based on previous work, this study is to explore the role of Wnt/ β-catenin/Tcf4 signaling pathway in the course of differentiation of the human corneal epithelial cells(HCECs) induced by the embryonic stem cell microenvironment. The key factors in Wnt signaling, β-catenin, Tcf4 and so on are going to be evaluated at both mRNA and protein levels in HCECs by real-time PCR, Western blotting, immunostaining and RNA interference. This project will clarify the regulation functions of this signaling pathway in the mechanism of the adult cells dedifferentiation via ES micro-environment and reveal the essence of the ES-micro-environment. This study may have high impact and clinic implication on the expansion of corneal epithelial cells in regenerative medicine, especially for ocular surface reconstruction.

细胞微环境对干细胞命运起重要的调控作用。胚胎干细胞微环境可使成体细胞"去分化"，重获干细胞表型及强增殖能力，然而其发生机制，尤其是细胞信号通路在其间的调控机制仍不清楚。Wnt信号途径是细胞生长、发育和分化的关键途径。我们前期利用胚胎干细胞微环境培养体系诱导角膜上皮细胞去分化，并已发现Wnt/integrinβ1-FAK-Akt信号通路的激活参与调控该过程。在此基础上，本项目拟利用免疫印迹、RT-PCR、Western blot、RNA干扰等技术，在mRNA和蛋白水平，研究与细胞增殖和分化更为密切相关的Wnt/β-catenin/Tcf4信号通路中的关键因子在胚胎干细胞微环境作用下的角膜上皮细胞内表达水平的变化，阐明该信号通路在其间的调控机制，从而揭示胚胎干细胞微环境作用的本质。这将对再生医学大量扩增细胞,尤其是获得用于进行眼表重建的角膜上皮细胞具有重要而深远的临床意义。

胚胎干细胞微环境具有延缓体细胞衰老，促进其增殖的作用。本研究进一步验证和完善了我们提出的ES微环境通过激活Wnt/integrinβ1- FAK- Akt信号通路加强共培养细胞干性的假设。通过检测在胚胎干细胞微环境下扩增的角膜上皮细胞中干细胞标志物表达水平及与细胞增殖和分化密切相关的Wnt/β-cateni/Survivin/Tcf4 信号通路活化情况，验证了其与TCF4及FAK、Wnt信号通路的相关性；利用siRNA技术阻断Tcf4的表达，检测siRNA干扰前后FAK/Wnt信号通路与p21蛋白的表达情况，发现ESC-CM培养体系促进角膜缘上皮干细胞特性的维持可能是通过P63及细胞周期相关蛋白P21及Wnt/β-catenin/Tcf4信号通路的激活等实现的。这对大量扩增并获得可用于临床眼表重建的角膜上皮细胞具有重要的意义。