微纳米颗粒复合组织工程化角膜上皮膜片重建角膜眼表的机制及疗效研究

A clear cornea is essential to visual acuity and depend on stromal avascularity and epithelial integrity. Corneal renewal and repair are mediated by stem cells of the limbus. Many ocular diseases may destroy the limbus, causing limbal stem cell deficiency (LSCD). The invasion of bulbar conjunctival cells leads to neovascularization, chronic inflammation, and stromal scarring, with corneal opacity even loss of vision. Allogeneic corneal transplantation restores transparency temporarily, but eventually, the conjunctival cells begin to invade and resurface the cornea. The only way to prevent this invasion is to restore the limbus. In clinical experience, allogeneic limbus transplantation application has long been limited by transplantation immunological rejection, however, restoration through the grafting of limbal fragments obtained from the uninjured eye usually seems unrealistic for the insufficient healthy limbal tissue. Remarkable advances in the understanding of external signals required for the maintenance of adult stem cell properties and nanotech tissue engineering have occurred in recent years, providing possibilities to restore limbus with ex vivo tissue engineered corneal epithelium..The current study is to screen key genes that maintain limbal stem cells (LSC) biological functions in cynomolgus monkey with RNA-sequencing. We use autologous cynomolgus monkey limbal stem cells as seed cells and degradable Hystem-C hydrogel as scaffold, composited with nanoparticles contained key gene modifiers to maintain LSC biological functions. The construction of new type of tissue engineered corneal epithelium would be applied to alkali burns of cynomolgus monkey corneas. Clinical and histological examinations would be carried out to evaluate the efficacy and mechanism of corneal reconstruction. This study improve current clinical strategies of LSCD, providing a new source of LSC transplantation.

各种因素引起的角膜缘干细胞衰竭导致的重度眼表疾病一直是治疗的难点。目前广泛应用的异体角膜缘移植受限于免疫排斥反应，而自体角膜缘移植常由于残留健康组织过少而难以实现。近年来角膜缘干细胞信号通路的深入研究和纳米组织工程技术的兴起和发展为体外大量扩增角膜上皮细胞，构建组织工程角膜上皮膜片提供了可能性。本研究采用RNA-sequencing技术筛选维持角膜缘干细胞(LSC)生物学功能的关键因子，取食蟹猴自体LSC进行体外扩增作为种子细胞，用可降解Hystem-C水凝胶为组织工程细胞载体，结合具有缓释功能的微纳米颗粒包裹关键因子调节剂维持LSC的生物学功能，构建新型角膜上皮膜片，重建食蟹猴碱烧伤模型的角膜眼表，术后观察临床修复效果并进行动态分子病理学检测，评估组织工程化角膜上皮膜片重建角膜眼表的术后疗效及内在机制。本研究优化了目前的LSC衰竭眼表疾病的治疗模式，为角膜眼表重建提供了较好的材料来源。

角膜盲是我国的第二大致盲眼病，其中由于各种因素引起的角膜缘干细胞衰竭导致的重度眼表疾病一直是治疗的难点，近年来角膜缘干细胞信号通路的深入研究和纳米组织工程技术的兴起和发展为体外大量扩增角膜上皮细胞，构建组织工程角膜上皮膜片提供了可能性。本课题组在面上项目基金资助下，采用单细胞测序技术筛选角膜缘干细胞(LSC)相关标志物，发现GATA2、IKZF1、SPI1有望成为新的角膜缘干细胞标志物，且已进行体外定位和功能验证，对体外纯化及分离人角膜缘干细胞具有重大科学意义和应用价值，也为人类基因表达谱、角膜缘干细胞及其微环境研究奠定基础；与此同时，取食蟹猴自体LSC进行体外扩增作为种子细胞，用可降解Hystem-C水凝胶为组织工程细胞载体，结合具有缓释功能的微纳米颗粒包裹关键因子调节剂维持LSC的生物学功能，构建新型角膜上皮膜片，重建食蟹猴碱烧伤模型的角膜眼表，术后观察临床修复效果，评估组织工程化角膜上皮膜片重建角膜眼表的术后疗效及内在机制。本研究优化了目前的角膜缘干细胞衰竭眼表疾病的治疗模式，应用组织工程技术体外扩增构建角膜上皮，为角膜病及眼表疾病的临床治疗提供较好的替代疗法。在该基金资助下培养了博士研究生2名，硕士研究生2名，课题组成员共发表SCI文章6篇，IF总计11.96。