WNT7A/FZD5调控角膜缘干细胞定向分化的分子机制研究

Limbal stem cell (LSC) is crucial to the maintenance of the function of corneal epithelium and corneal transparency. Abnormal differentiation of LSC due to various reasons causes the corneal surface to turn into an opaque keratinized skin-like epithelium, which leads to blindness in millions of people worldwide. As the main treatment, corneal transplantation faces severe donor deficiency, immune rejection risk and other issues. We have found in previous studies that PAX6 is a key transcription factor for corneal epithelial cell fate determination, and WNT7A/FZD5 acts as the upstream regulators. In current proposal, we intend to focus on the molecular mechanism of WNT7A/FZD5 in LSC lineage commitment, find specific targets for the control of LSC differentiation, and explore the feasibility of using small molecule compounds in the treatment of blindness caused by abnormal differentiation of LSC.

角膜缘干细胞 (Limbal Stem cell，LSC)对于维持角膜上皮功能和角膜透明发挥至关重要的作用。各种原因导致的 LSC异常分化常常表现为角膜上皮的皮肤样化生，角膜丧失透明性，是世界主要的致盲眼病。以角膜移植术为主要治疗手段面临供体严重匮乏和免疫排斥风险等问题。我们在前期研究中发现PAX6是角膜上皮细胞命运决定的关键转录因子，而WNT7A/FZD5为其上游调控信号。本项目拟围绕WNT7A/FZD5调控LSC定向分化的分子机理，找寻可调控LSC分化方向的特异性靶点，深入探讨应用小分子化合物等治疗LSC异常分化致盲眼病的可行性。

角膜的完整性和透明度对视觉功能至关重要。角膜上皮的自我更新和再生是由位于角膜缘区的基底层角膜缘干细胞（LSC）所维持的。各种慢性炎症、感染、Stevens–Johnson综合征和基因突变在内的各种损伤均可能导致LSC功能障碍，引起角膜混浊、新生血管和角膜结膜化为特征的角膜失明。首先，我们开发了一种无滋养层、无血清的LSC扩增培养体系，能够获得纯度及均一性高的LSC，用以研究调控LSC细胞命运决定的关键因子。通过整合超级增强子和染色质可及性，我们描绘了LSC的核心转录调控环路（CRC），发现WNT7A、RUNX1和SMAD3是维持角膜上皮特性和稳态维持所必需的超级增强子。RUNX1、FOXC1或SMAD3缺失都将抑制PAX6并诱导LSC分化为皮肤样上皮细胞。RUNX1、PAX6和SMAD3（RPS）相互作用并协同建立CRC以控制谱系特异性顺式调节图谱。RPS缺陷将导致角膜上皮细胞特性转换和功能障碍，并与多种人类角膜疾病密切相关。我们的工作指明超级增强子在干细胞特性维持和谱系承诺方面的协同作用，为复层上皮稳态和发病机制提供了全面的调控原则，并为角膜失明患者提供了潜在的治疗靶点。