EX VIVO EXPANSION OF LIMBAL EPITHELIAL STEM CELLS

角膜上皮的动态平衡最终是通过 角膜缘上皮干细胞的增殖分化定位 在角膜和结膜之间。许多人类角膜疾病都有 现在发现由于角膜缘干细胞的破坏而表现出角膜缘干细胞缺乏 角膜缘干细胞群体或角膜缘基质功能障碍。患有疾病的患者 角膜缘缺乏症有严重的视力丧失和烦人的眼睛 不适，是传统角膜移植的不佳选择。 自体角膜缘干细胞移植已被用于 恢复正常的角膜上皮和视力。这样做的主要缺点是 手术程序是将供体眼球摘除1/3 一半的正常角膜缘可能最终会发展为角膜缘缺乏症。这个 进展报告显示，羊膜移植作为底物 帮助修复未发炎的角膜缘基质，并扩大剩余的角膜缘干细胞 人口。这一结果提示了一种假设，即角膜缘干细胞种群可能是 小范围角膜缘活检羊膜培养扩增(约1mm~3) 使用羊膜培养(目标1)。这一文化体系将允许 角膜缘缺乏症的发病机制探讨。具体来说，他们将 检查基底膜在支持角膜缘干细胞扩张中的作用 (目标2)，炎症的致病作用是导致角膜缘缺乏 通过分析炎性细胞因子如何通过以下途径降解基底膜 上调基质金属蛋白酶(MMPs)(目标3)。最后，他们会 验证这种新方法治疗兔角膜缘的临床前疗效 体内缺乏(目标4)。完成这些目标将允许使用这一新的 新型角膜缘干细胞在羊膜上的体外扩增策略 消除目前自体角膜缘移植问题的外科手术。 此外，由此产生的新知识也将有助于开发新的 预防角膜缘缺乏症的治疗，为微笑铺平道路 以结膜表面为目的的结膜上皮手法 未来的重建。上皮性干细胞的体外扩增也是一种 针对干细胞水平和组织的基因治疗的第一步 用于未来治疗和重建的上皮组织工程学。

Homeostasis of the corneal epithelium is ultimately achieved by proliferation and differentiation of limbal epithelial stem cells (SC) located between the cornea and the conjunctiva. A number of human corneal diseases have now been found to exhibit limbal SC deficiency as a result of destruction of the limbal SC population or dysfunction of the limbal stroma. Patients with limbal deficiency suffer from severe visual loss and annoying ocular discomfort, and are poor candidates for conventional corneal transplantion. Transplantation of autologous limbal SC (limbal autograft) has been used to restore the normal corneal epithelium and vision. The major drawback of this surgical procedure is that the donor eye with the removal of one third to one half of the normal limbus may eventually develop limbal deficiency. The progress report shows that transplantation of amniotic membrane as a substrate helps restore a non-inflamed limbal stroma and expand the remaining limbal SC population. This result prompts the hypothesis that limbal SC population can be expanded on amniotic membrane cultures from a small limbal biopsy ( about1 mm3) using amniotic membrane cultures (Aim 1). This culture system will allow exploration of the pathogenesis of limbal deficiency. Specifically, they will examine the role of the basement membrane in supporting limbal SC expansion (Aim 2), and the pathogenic role of inflammation is causing limbal deficiency by analyzing how inflammatory cytokines may degrade the basement membrane by upregulating matrix metalloproteinases (MMPs) (Aim 3). Finally, they will verify the pre-clinical efficacy of this new approach in treating rabbit limbal deficiency in vivo (Aim 4). Completion of these aims will allow use of this new strategy of ex vivo expansion of limbal SC on amniotic membrane as a new surgical procedure to eliminate current problems of limbal autograft. Furthermore, the new knowledge thus generated will also help to develop a new therapy for preventing limbal deficiency, and pave the way for smilar manipulations of conjunctival SC for the purpose of conjunctival surface reconstruction in the future. Ex vivo expansion of epithelial SC is also a first step for gene therapy targeted at the SC level, and for tissue engineering of an epithelial tissue for future therapeutics and reconstruction.