单个角膜缘干细胞培养构建可移植性角膜缘功能单元

Cornea disease accounts for the major blindness in the world, among which limbal stem cell deficiency (LSCD) is the main risk factor that prevent these patients from restoring sight. Limbal stem cell(LSC) refers to stem cells that could self-renewal and differentiation into cornea epithelium, LSC transplantation is an effective way to treat LSCD, but LSC has not been cultured and characterized at single cell level yet. Previously, Dispase digestion method has been used as the standard method to isolate "LSC", but that maybe not the fact. We have demonstrated that Collagenase digestion could isolate a group of progenitor cells from human limbus that have better stemness features than Dispase, we also isolated a group of supporting cell from human limbus microenvironment (niche), named limbal niche cell(LNC), which played key role in maintaining the stemness of epithelial progenitor cell isolated from limbus. In this project, we are going to mimic the limbal microenvironment based on LNC and corresponding culture condition, to isolate and culture LSC from human limbus,to rebuild transplantable limbal functional unit(TLFU) from a single stem cell, such a stem cell bank could provide stable source for clinic use to treat LSCD patients; the most active LSC clone sould be find out using a dividing and retro-judging strategy. The carry out of this project could set up the platform to characterize,culture and utilize LSC at single cell level.

角膜病是主要的致盲性眼病，其中角膜缘干细胞缺乏（LSCD）是阻碍角膜病患者复明的首位危险因素。角膜缘干细胞（LSC）是具有自我更新并定向分化为角膜上皮细胞功能的成体干细胞，体外培养的LSC移植是治疗LSCD的有效方法，但迄今为止，单个LSC细胞分离、鉴定方法尚不清楚。以往研究采用Dispase酶消化被认为是分离培养LSC的"金标准"，但事实并非如此。课题组前期研究发现采用Collagenase酶消化方法能获得在功能上优于以往方法分离培养的"LSC"，并发现LSC微环境中关键的支持细胞- - 角膜缘微环境细胞（LNC）。本课题拟在上述基础上优化培养条件，分离扩增LSC，构建可移植性角膜缘功能单元（TLFU）的干细胞银行，为临床治疗LSCD提供源源不断的供体。再采用机械分离一分为二的策略，逆向推断锁定最佳的"LSC"克隆，为最终从单个细胞水平分离、鉴定、扩增、应用LSC奠定理论和实验基础。

本课题试图模拟人角膜缘微环境，培养扩增LSC细胞克隆，建立从单个LSC体外培养构建TLFU的方法平台，筛选出真正的LSC细胞克隆，并从单个细胞水平进行初步分析。研究结果实验证明在体外及体内环境下，LNC细胞支持LSC均优于BMMSC，LNC移植治疗LSCD优于BMMSC； LNC细胞表达SCF、Nestin、REX1、SSEA4、PDGFRβ、P75NTR、MSC干细胞标志物CD73、CD90、CD105水平均高于BMMSC。这提示LNC是体外模拟LSC微环境的关键因素之一。基因芯片发现二者表达差异大于2倍的基因有2661个，主要在 WNT信号通路、细胞膜组成成分、细胞外基质形成方面存在差异。LNC表达Laminin高于BMMSC，而表达Collgagen IV低于BMMSC，这些差异可能是LNC细胞支持LSC细胞的重要通路。体外获得的LSC克隆接种于羊膜表面构建TLFU需要同时接种LNC。LSC克隆细胞与非LSC细胞克隆的差别除了体积较小，还可能与其表达胚胎干细胞抗原Oct4、Sox2 、NANOG、 SSEA4、ABCG2，干细胞因子SCF及其配体KIT有关。