Tcf4/Wnt信号系统调控皮肤细胞重编程重建角膜上皮的研究

With support of National Natural Science Foundation of China (30901634), during the last three years we devoted ourselves into research about the phenotype and properties of corneal epithelial stem cells, and ocular surface reconstruction with induced pluripotent stem cells. We found that transcription factor Tcf4, the key factor of Wnt signaling pathway, could maintain the properties of corneal epithelial stem cells, which keep a less differentiated phenotype and a high proliferative capacity (Lu R, et al. Stem cells, 2012) (Lu R, et al. IJBCB, 2011). However, the important downstream factors of Tcf4/Wnt signaling pathway are still not clear and the underlying mechanism by which these properties of corneal epithelial stem cells are maintained has not been well elucidated. Moreover, we observed epidermal stem cells have the properties of reprogramming. Under ocular surface niche, epidermal stem cells reprogrammed into conjunctiva-like epithelial cells (Lu R, et al. Plos One, 2011). However, the reprogramming properties of epidermal stem cells are limited and the reprogramming efficiency is very low. Recent evidence suggests that the non-cell autonomous reprogramming is not unidirectional. So under proper niche, epidermal stem cells could de-differentiate into pluripotent stem cells. . The current study is to induce epidermal stem cells reprogramming into pluripotent stem cells by non-cell autonomous recruitment of endogenous OKSM factors. With ES cell conditioned medium as niche in vitro, epidermal stem cells could be induced into pluripotent stem cells. This new approach avoids the disadvantages associated with the use of viral vectors included the risk of genomic integration and insertional mutagenesis. At the same time, with microarray analysis, we would identify several key factors of Tcf4/Wnt signaling pathway. The iPSCs are knocked in/ knocked out with these selected genes. And then gene modifier iPSCs were transplanted into the mouse eyes with corneal alkali burn. With the animal experiments, it would be elucidated how the underlying mechanism of Tcf4/Wnt signaling pathway regulates the corneal epithelial stem cells.

青年基金资助下，进行了角膜缘干细胞分子调控和诱导性多功能干细胞（iPSC）重建眼表研究。发现，Tcf4/Wnt信号通路对维持角膜缘干细胞特性有重要作用(Stem Cells; IJBCB)，但通路中关键因子尚未明确，细胞调控机制也未阐明；此外皮肤干细胞有重编程能力，在结膜损伤眼表微环境中，重编程为结膜上皮样细胞(Plos One)。然而皮肤干细胞多向分化潜能有限，重编程效率极低。但若给予适宜微环境，可能使其去分化为有多向分化潜能的iPSC。因此，本研究采用非细胞自主性重编程，改变细胞微环境，诱导皮肤干细胞重编程为iPSC，避免传统外源基因插入带来的生物安全问题，获得一种高效安全的iPSC。同时，利用基因芯片技术筛选Tcf4/Wnt信号通路中对角膜缘干细胞特性维持起关键作用的基因，基因修饰iPSC，在小鼠角膜碱烧伤模型体内揭示Tcf4/Wnt信号通路关键因子对干细胞分化重建角膜上皮的调控机制

各种因素引起的角膜缘干细胞衰竭导致的重度眼表疾病一直是治疗的难点。目前广泛应用的异体角膜缘移植受限于免疫排斥反应，而自体角膜缘移植常由于残留健康组织过少而难以实现。Tcf4/Wnt信号通路对维持角膜缘干细胞特性有重要作用，但通路中关键因子尚未明确，细胞调控机制也未阐明；此外皮肤干细胞有重编程能力，在结膜损伤眼表微环境中，重编程为结膜上皮样细胞。本研究在执行先前的计划要点的同时，也对Tcf4/Wnt信号通路对眼表干细胞的作用进行了研究，其研究结果主要分为以下四方面：1）检测人角膜缘干细胞（LSC）的基因表达谱研究为进一步非人灵长类动物的基因表达谱；2）研究Tcf4在人角膜缘干细胞（HLSC）的生物学特性中的作用；3）Tcf4在结膜上皮组织的表达与结膜上皮干细胞的分布；4）Tcf4对结膜上皮细胞增殖特性的作用。旨在进一步阐明Tcf4/Wnt信号通路在维持眼表干细胞特性的具体机制，为寻找可能的重建眼表材料靶点提供新方向。研究成果发表SCI论文4篇，于2015年以第一完成人身份主持的项目《干细胞与眼表重建的系列研究》（A04-0-3-02）获得广东省科学技术三等奖，4次在国内外眼科学术会议上进行交流，培养毕业硕士研究生四名，在读硕士研究生四名。