转分化口腔黏膜上皮干细胞增强其在眼表重建中的抗新生血管作用

Limbal stem cell deficiency (LSCD) and corneal neovascularization are leading causes of blinding corneal disease. Allogeneic cornea and limbal stem cell transplantation has become a promising therapy for LSCD to reconstruct the ocular surface and restore the vision. However, the shortage of donor corneas and reject reaction constrain its application in China. Nowadays, cultivated oral mucosal epithelial cells transplantation has shown encouraging results in reconstructing ocular surface affected by LSCD, in which 3T3 cells are widely used in co-culturing the oral epithelium. However, it takes the risk of transmitting murine-derived diseases to human. Moreover, long-term outcomes were less satisfactory due to a high rate of peripheral corneal neovascularization. In contrast, limbal epithelial cells have anti-angiogenic activity in vivo and in vitro. Since the fate decision of stem cells is regulated by a certain niche, herein, this study was conducted to mimic limbal niche environment to transdifferentiate oral mucosal epithelial stem cells into corneal epithelial-like cells in vitro in order to strengthen the anti-angiogenic activity and avoid the usage of 3T3 feeder cells. In the present study, we will analyze the anti-angiogenic function and mechanism of these epithelial sheets derived from different feeder cells. This novel study can be used for investigating how oral mucosal epithelial stem cells fate decision might be regulated in the limbal niche, the mechanism of anti-angiogenesis and how to improve the outcome of LSCD treatment.

角膜缘上皮干细胞缺陷及新生血管是大多数致盲角膜病的最主要原因。同种异体角膜和干细胞移植为重建眼表的重要手段，但我国角膜供体严重匮乏及异体排斥反应制约了其在该领域的应用。目前，体外培养的自体口腔黏膜上皮在临床眼表重建中取得成功。但是，应用鼠胚胎来源的成纤维细胞作为滋养层，有传播未知鼠源性疾病的风险；且在长期随访中，发现不同程度的周边角膜新生血管化。角膜缘上皮细胞具有抗新生血管作用，干细胞的微环境决定干细胞的分化命运。因此，本课题拟体外模拟角膜缘基质微环境，转分化口腔黏膜上皮干细胞为角膜缘上皮（样）细胞，增强其抗新生血管的能力；同时，拟对比分析不同条件下培养的角膜缘上皮与口腔黏膜上皮在体外实验及动物体内实验血管新生中的作用及机制。本研究采用自体组织细胞可克服采用鼠来源细胞的缺陷，同时对于了解干细胞调控及其抗新生血管的的机制，改善角膜缘干细胞缺陷的治疗效果具有重要理论价值和实际意义。

角膜缘上皮干细胞缺陷及新生血管是大多数致盲角膜病的最主要原因。同种异体角膜和干细胞为重建眼表的重要手段，但我国角膜供体严重匮乏制约了其在该领域的应用。目前，体外培养的自体口腔黏膜上皮在临床眼表重建中取得成功。但是，应用鼠胚胎来源的成纤维细胞作为滋养层，有传播未知鼠源性疾病的风险；且在长期随访中，发现不同程度的周边角膜新生血管化。角膜缘基质微环境细胞作为角膜缘微环境中的最重要一环，对干细胞分化的方向起到了关键性的作用。前期研究也证实成体干细胞在角膜缘微环境中可以向角膜上皮样细胞分化。本课题组对角膜缘基质微环境细胞进行了深入研究，成功分离了大鼠口腔黏膜上皮干细胞，建立起角膜缘基质微环境细胞与口腔黏膜上皮细胞的三维及transwell小室共培养体系，定量分析了经过共培养的大鼠口腔黏膜上皮干细胞向角膜上皮样细胞转化的趋势及抗新生血管的能力，并通过大鼠角膜缘干细胞损伤模型说明转分化后的口腔黏膜上皮干细胞抗新生血管及眼表重建的能力。研究结果总结如下：通过免疫荧光染色确定了口腔黏膜上皮细胞和角膜缘上皮细胞表达了共同的分化标志物CK3和共同的干细胞标志物△Np63a，而CK12和Pax6只表达于角膜上皮层，是角膜上皮特异性指标；用dispase消化角膜缘组织得到角膜缘上皮细胞，采用最优的胶原酶消化法得到LNC；用dispase消化口腔黏膜可以得到口腔黏膜上皮干细胞，其在3dmatrigel条件及transwell下均可促进口腔黏膜上皮细胞转化为表达CK12及Pax6的角膜上皮样细胞；PCR及western证实，我们通过不断更新滋养的LNC细胞，以3T3细胞作为参照，能在一定程度上提高转化的效率并且上调了抗新生血管因子PEDF，sFlt-1的表达，下调了血管生成因子VEGF的表达；口腔黏膜上皮细胞可以在人工角膜基质上良好生长；动物实验说明转分化口腔黏膜上皮细胞有助于眼表的恢复。本课题通过建立体外口腔黏膜上皮与LNC的共培养体系，利用转化医学，培养出了表达角膜特异性指标并且低血管生长因子的角膜样上皮，对于治疗角膜缘干细胞缺乏及眼表重建具有重要的意义。