组织工程角膜三维构建的实验研究

目的以人胚胎干细胞（hESC）诱导细胞为种子细胞，以脱细胞猪角膜基质（APCM）为支架三维构建生物工程角膜，以期用于穿透性角膜移植，解决角膜供体极度匮乏的难题。方法实验研究。无菌条件下将新鲜猪角膜组织置于0.5% SDS溶液中4 ℃脱细胞 24 h，获取APCM。将hESCs与人角膜基质细胞通过Transwell共培养5 d，获取眼周间充质干细胞（POMPs），再于人晶状体上皮细胞源性条件培养基继续培养14 d获取角膜内皮样细胞并进行鉴定和筛选纯化。将纯化后扩增的角膜内皮样细胞接种于APCM构建角膜内皮植片，并移植入角膜内皮功能失代偿动物模型进行泵功能评估；采用人角膜缘干细胞（LSCs）来源的条件培养基培养hESCs 12 d，诱导其分化人角膜上皮样细胞并筛选鉴定，将其与APCM构建的角膜上皮植片移植于LSC失代偿动物模型的角膜缘，观察其眼表修复能力。结果诱导的人角膜内皮样细胞表达内皮细胞相关标记物vimentin、N-cadherin、Na+/K+ATP酶和ZO-1。构建的角膜内皮植片能够促使角膜内皮功能失代偿动物的角膜逐渐恢复透明。构建的角膜上皮细胞植片具有4～5层细胞复层结构，类似于正常角膜上皮，且能够一定程度上修复LSC失代偿动物模型眼表。结论采用hESCs诱导分化来源的细胞与APCM构建的人角膜内皮植片和人角膜上皮植片具有类似于正常角膜的功能，为全层生物角膜的构建提供了良好的实验和理论基础，具有良好的临床应用前景。

Three-dimensional construction of tissue-engineered cornea in vitro

Objective To develop a corneal replacement for penetrating keratoplasty (PKP).A three-dimentional,tissue-engineered cornea is constructed by using human embryonic stem cells (hESCs) derived corneal endothelial-like cells and corneal epithelial-like cells as seed cells,and acellular porcine cornea matrix (APCM) as scaffold.Methods Experimental study.To obtain APCM,sterile fresh porcine cornea was decellularized with a 0.5％ SDS solution for 24 hours at 4 ℃ and then washed 8 times with PBS over 24 hours to remove SDS.To obtain corneal endothelial-like cells derived from hESCs,periocular mesenchymal precursors (POMPs) were developed first by using a transwell co-culture system of hESCs with human corneal stromal cells,and then culturing in a lens epithelial cell-conditioned medium for 14 days.The isolated corneal endothelial-like cells were expanded,labeled and seeded onto APCM lamellas to construct the corneal endothelial-like cell sheets.Pump functions of the cell sheets were measured by the corneal endothelium transplantation of rabbit corneal endothelium dysfunction models in vivo.Corneal epithelial-like cells derived from hESCs were cultured with an LSC-conditioned medium,and then seeded on APCM to construct corneal epithelial-like cell sheet for limbal stem cells deficiency model transplantation.Animals were followed up with a weekly slit lamp microscope exam and HE staining was performed after surgery.Results The induced cells expressed corneal endothelial cells related markers such as Na+/K+ATPase alpha1,ZO-1,N-cadherin and vimentin.The constructed corneal endothelial-like cell sheets improved the corneal transparency of rabbit cornea endothelium in dysfunction models.Corneal epithelial-like cells formed 4 to 5 layers of cells on APCM and possessed the potential to repair a damaged ocular surface in vivo.Conclusion The construct derived from hESCs induced cells and APCM possessed the similar function and structure of a natural cornea,and might be used for PKP in future.