角膜内皮功能失代偿是眼科临床的常见病，却是难治性疾病。异体角膜移植（包括内皮移植）受到了供体来源和免疫排斥的两大限制，因此寻找自体来源丰富的多潜能干细胞替代损伤的角膜内皮细胞是解决问题的关键。我们在前期的研究中发现，通过体外和体内微环境的诱导，可将内皮祖细胞分化为角膜内皮样细胞并在动物模型中具有修复内皮层缺损的能力。但在细胞培养诱导过程中存在细胞干性丢失、诱导分化关键调控基因、调控分化的信号通路不明确等问题。针对这些问题，本课题拟进一步开展以下研究：应用3D培养技术维持自体来源骨髓内皮祖细胞干性；使用CRISPR技术上调内皮祖细胞PAX6基因的表达，研究其在诱导分化过程中的作用；以及N-cad—p120—RhoA接触抑制相关信号通路在诱导分化中的作用机制。研究的成功将为明确多潜能干细胞诱导分化为角膜内皮细胞的机制提供新的理论与实验支持，并最终有望真正实现自体干细胞移植治疗角膜内皮失代偿。

Corneal endothelial decompensation is a common but refractory disease in ophthalmology. Cornea allograft transplantation including endothelial keratoplasty has two limits, source of donor and immune rejection. Therefore, looking for a rich source of autologous pluripotent stem cell to replace the damaged corneal endothelium is the key to solve the problem. In our previous study, we found that endothelial progenitor cells (EPCs) induced by microenviroment in vitro and vivo can differentiate into corneal endothelial like cells and repair the corneal endothelium defect in animal models. But EPCs lost its stemness gradually during cell culture, the key genes in cell differentiation is unkown, the signaling pathway in cell differentiation is be to found. Aiming to solve these problems, our team intend to carry out the following research: application of 3D cell culture technology to maintain stemness of endothelial progenitor cells; up-regulation of PAX6 gene by CRISPR system to study its role in the process of cell differentiation; and then to study the mechanism of N-cad - p120 - RhoA contact inhibition related signaling pathway in EPCs’ differentiation. The success of the research will provide new theoretical and experimental support for the mechanism of multipotential stem cells differentiate into corneal endothelial cell. It is expected to achieve the really autologous stem cell transplantation in the treatment of corneal endothelial decompensation.