微环境和Rho/ROCK通路共同作用内皮祖细胞修复角膜内皮层的研究

Corneal endothelial dysfunction caused by various diseases is very difficult to treat in clinics, 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 bone marrow-derived endothelial progenitor cells can be induced into corneal endothelial like cells through co-culture system with HCECs. And based on the theory that cell niche controls the differentiation of stem cells, Rho/ROCK signal is contribute to cell barrier.We intend to undertake the following studies: Using immunomagnetic beads to label and purify stem cells. Endothelial progenitor cells will be induced in vitro by co-culture with CEC and epithelial cells of lens.Then the induced progenitor cells will be injected into anterior chamber of rabbits, and then be oriented to attach on the surface of corneal Descemet's membrane by magnetic field outside. Thus, the feasibility of endothelial progenitor cells being induced to corneal endothelial cells in specific niche can be studied. At the same time, the role and mechanism of Rho/ROCK signaling pathway on enhancing the barrier of endothelial progenitor cells will be studied as well. The success of the study is expected to seek a real alternative of autologous stem cells to replace corneal endothelial cells. The advantages not only lie in the sufficient source of cells, but also that immune rejection can be avoided, and this method can overcome the limitations of biological materials. The results of this study can also provide new ideas and method for the clinical treatment of corneal endothelial dysfunction.

角膜内皮功能失代偿导致角膜混浊和视力下降是临床治疗的难题，角膜内皮细胞增殖能力非常有限，寻找自体来源丰富的多潜能干细胞移植是解决这一难题的有效方法。我们在前期研究中初步证实了骨髓内皮祖细胞在体外可诱导分化为角膜内皮样细胞并在猫眼中具有一定的修复角膜内皮缺损的能力，但其诱导条件和治疗效果尚需进一步探讨。为此我们拟结合niche微环境调控干细胞分化的理论，以及Rho/ROCK通路与细胞屏障作用的关系，深入研究：免疫磁珠提纯内皮祖细胞，通过角膜内皮和晶状体上皮细胞共培养体外诱导，前房注射诱导后的祖细胞至角膜内皮微环境中，探讨体外诱导加体内微环境作用后其分化再生修复角膜内皮的能力，同时加入促进形成细胞屏障作用的Rho/ROCK通路抑制剂，以达到完全修复内皮层的作用。研究结果有望真正实现自体干细胞移植代替角膜内皮细胞，不仅来源充足，而且避免了免疫排斥反应，为临床治疗角膜内皮失代偿疾病提供新的方法。

角膜内皮功能失代偿导致角膜混浊和视力下降是临床治疗的难题，利用干细胞的组织工程修复受损的角膜内皮层是国内外研究的方向。本项目按照研究计划，分离培养角膜内皮生长微环境中存在的各种细胞，并制备条件培养液，对内皮祖细胞进行诱导分化，并对其诱导效果及机制展开一定的研究。同时，项目组成员根据角膜位于眼部表层，角膜透明便于注射操作等特点，改进了前房细胞注射方法，设计磁珠连接干细胞，利用磁场牵拉，使修复的干细胞能顺利达到角膜内皮层，起到定向修复的作用。为内皮祖细胞向角膜内皮细胞诱导分化研究和干细胞前房注射治疗打下理论和实验基础。角膜内皮细胞体外培养增殖差是制约角膜内皮层修复基础研究的困难之一，课题组成员在本项目的支持下，开展了干细胞条件培养液促进角膜内皮细胞体外增殖的相关研究，为后续开展角膜内皮层修复提供实验基础。此外课题组成员还开展了其他眼表角膜修复相关的研究，包括角膜内皮细胞增殖相关信号通路的基础研究；结膜组织修复的基础研究；十型眶面裂相关临床研究等等。项目组成员按照计划完成项目研究，共发表标注论文12篇，获得专利1项，培养研究生5名，顺利达到项目预期。