Niche细胞源exosomes诱导口腔粘膜上皮干细胞构建功能性角膜上皮层的研究

Cultured oral mucosal epithelial transplantation(COMET) has been used as a treatment for bilateral limbal stem cell deficiency for more than ten years. The biggest drawback to COMET is corneal vascularization in long-term follow-ups. Oral mucosal epithelial cells cannot completely substitute and implement corneal epithelium’s function mainly because of its phenotypic differences from corneal epithelial cells. Our preliminary studies showed that niche cells derived exosomes (NC-Exos) could induce oral mucosal epithelial stem cells (OMESCs) to express Pax6, and to express the corneal epithelium marker--K12 and an anti-angiogenic factor--sVEGFR-1 when differentiating. These results indicate that the induced OMESCs have the potential to reconstruct the corneal epithelium functionally. Currently, it is urgent to study and investigate the inducing conditions, therapeutic effects, and mechanisms further. Considering that, we intend to select the best NC-Exos inducing conditions, to study how the induction would influence the anti-angiogenic, barrier, defending and repairing functions of tissue-engineered epithelium layers, and then to verify these functions by animal experiments. On the other hand, we will further investigate the sVEGFR-1 expression mechanism by screening the NC-Exos miRNA expression profiling and functional testing. The success of the study is expected to solve the problem of corneal vascularization after COMET in the long terms.

口腔粘膜上皮细胞移植（COMET）用于临床治疗双眼角膜缘干细胞衰竭已有10余年历史，最大的不足是移植后远期角膜血管化。口腔粘膜上皮细胞与角膜上皮细胞表型上的差异使得它不能从功能上完全替代角膜上皮。我们前期研究发现，niche细胞源exosomes（NC-Exos）可诱导口腔粘膜上皮干细胞（OMESCs）表达Pax6，并分化表达角膜上皮标志物K12和抗血管化物质sVEGFR-1，提示OMESCs诱导后具有功能性修复角膜上皮层的潜力。目前，诱导条件、治疗效果和诱导机制亟待进一步研究和探索。因此，本课题拟筛选NC-Exos诱导OMESCs的最优方案，研究诱导对OMESCs构建组织工程上皮层的抗血管化能力和屏障、防御、修复能力的影响，并进行动物实验验证其功能；通过筛选NC-Exos中特异表达的miRNA，深入研究其调控sVEGFR-1表达的机制。研究的成功有望解决COMET远期角膜血管化的难题。

角膜缘干细胞衰竭是最常见的角膜及眼表疾病之一，也是我国及世界上最主要的致盲眼病之一。组织工程角膜上皮重建技术通过体外种子细胞扩增环节可以极大的减少医者对组织取材量的需求，因而组织工程角膜上皮重建技术是应对角膜供体有限和自体供区不足的利器。口腔黏膜上皮细胞是目前临床最常用于替代角膜缘干细胞的自体种子细胞，但因其与角膜上皮细胞表型不同，移植后远期效果不佳。课题组成员建立和完善了角膜缘微环境外泌体诱导口腔黏膜上皮干细胞向角膜上皮方向分化的体系；并进一步在器官培养模型中研究眼表微环境对口腔黏膜上皮细胞的诱导作用以及组织工程口腔黏膜复层上皮层的屏障功能。在此基础上，课题组于大鼠角膜碱烧伤模型和角膜缘干细胞衰竭患眼中验证了口腔黏膜上皮移植在角膜上皮层重建中的作用，并探讨了微环境诱导口腔黏膜上皮细胞转分化的机制，初步阐明微环境诱导的Pax6基因表达对口腔黏膜上皮向角膜上皮方向转分化有一定正向促进作用。本课题的研究，为今后临床应用转分化的口腔黏膜上皮眼表重建提供了实验基础，为角膜缘干细胞衰竭的治疗提供了新思路和新方法。