干细胞微环境及Dkk2-Wnt/beta-catenin信号通路调控内皮祖细胞向角膜内皮分化的研究

Corneal endothelial dysfunction caused by various diseases is very difficult to treat in clinical ophthalmology, resulting in corneal opacity and visual loss. The proliferation of Human Corneal Endothelial Cells (HCEC) is limited both in vivo and in vitro. Therefore, the key solution to the problem would be replacing the excessively lost HCECs with transplanted pluripotent stem cells which have rich sources. In the preliminary studies, we found that endothelial progenitor cells（EPCs） can be planted into corneal endothelium to repair the endothelium defect. And based on the theory that cell niche controls the fate of stem cells,Dkk2 protein and Wnt signal plays an anessential role in corneal endothelial-liked cell differentation.We intend to undertake the following studies: Endothelial progenitor cells will cultured on adipose-derived stem cell extracellular matrix(ADSC-ECM) to promote the stemness of the EPCs. Dkk2 protein will added in culture medium of EPCs and the change of Wnt/β-catenin signaling will be observed. The relationship between EPC differentation and Wnt/beta-catenin signaling will be well studied . The success of the study is expected to seek a real alternative of autologous stem cell to replace corneal endothelial cells. The advantages not only lie in the sufficient source of cells, but also that immune rejection is avoided, This study can provide new ideas and method for the clinical treatment of corneal endothelial dysfunction.And the results will let us know more about involved cell signaling in stem cell differentaiton.

角膜内皮功能失代偿导致角膜混浊和视力下降，是眼科临床治疗的难题。角膜内皮细胞在体内外增殖能力非常有限，因此寻找自体来源丰富的多潜能干细胞移植以代替损伤的角膜内皮细胞是解决问题的关键。我们在前期研究中初步证实了内皮祖细胞有在动物模型中修复角膜内皮层缺损的能力。但存在内皮祖细胞体外培养干性丢失快；体外诱导方法及相关通路不明确等问题。根据干细胞微环境调控理论和Dkk2蛋白激活/抑制Wnt通路调控角膜内皮方向分化的理论，本课题拟开展以下研究：使用脂肪干细胞的细胞外基质维持内皮祖细胞在体外培养时的干性；在体外培养体系中加入Dkk2蛋白诱导内皮祖细胞向角膜内皮细胞方向分化，并研究Wnt/β-catenin通路在此诱导分化中的作用。研究的成功为干细胞向角膜内皮方向分化及其中涉及的细胞因子和信号通路变化提供理论和实验支持；为将来真正实现内皮祖细胞移植修复角膜内皮层治疗角膜内皮失代偿疾病提供新的思路和方法。

人角膜内皮细胞在体内无法增殖，各种原因造成的角膜内皮失代偿是常见角膜疾病之一，虽然可以使用角膜内皮层移植治疗，但国内角膜捐献供体缺乏，使角膜内皮失代偿的治疗成为临床治疗的难点。利用自身成体干细胞定向分化修复角膜内皮层是国内外研究的热点，但存在干细胞体外培养干性丢失快，条件培养液诱导分化效果不明确等难点。课题组成员建立和完善了脂肪干细胞细胞外基质培养内皮祖细胞的体外培养体系，能有效促进内皮祖细胞的增殖并能提高内皮祖细胞在体外培养时的干性。在随后的条件培养液体系中加入Dkk2因子，开展了Dkk-LRP5/6(Wnt/β-catenin)接触抑制相关通路在内皮祖细胞诱导分化为角膜内皮细胞中的作用机制的研究，初步说明了激活此通路与PAX6 蛋白表达及其他相关功能蛋白表达之间的关系。对今后内皮祖细胞的在体外扩增和干性维持提供实验基础，以及后续向角膜内皮细胞的精准分化调控，提供新思路和新方法。