组织工程角膜内皮的体外构建及在猕猴角膜移植中的应用  

Functions of tissue-engineered corneal endothelium in monkey corneal transplantations

作　　者：赵君[1] 樊廷俊[1] 

机构地区：[1]中国海洋大学海洋生命学院角膜组织工程实验室,青岛266003

出　　处：《中华眼视光学与视觉科学杂志》2016年第4期226-231,共6页Chinese Journal Of Optometry Ophthalmology And Visual Science

基　　金：国家高技术研究发展计划项目（2006AA02A132）;山东省重点研发计划项目（2015GSF121013）;山东省优秀中青年科学家科研奖励基金（BS2012YY040）;中央高校基本科研基金（201413008）

摘　　要：目的体外构建出形态结构功能正常的组织T程人角膜内皮（TE—HCE），并以猕猴动物模型验证其维持角膜透明的作用。方法实验研究。以非转染人角膜内皮单克隆细胞株细胞（mcHCE细胞）为种子细胞，以去上皮层修饰羊膜（mdAM）为载体支架体外构建mTE—HCE，对其透明度、形态、细胞密度、组织学结构、超微结构、功能蛋白的荧光表达进行检测；采用穿透性角膜移植手术（PKP）对猕猴右眼分别移植TE—HCE（3眼，TE—HCE组）和mdAM（3眼，mdAM组），未移植的左眼作为正常对照眼（6眼，对照组），利用裂隙灯显微镜、角膜内皮镜、角膜测厚仪和眼压计对术后动物的角膜透明度、角膜内皮细胞密度（ECD）、中央角膜厚度（CCT）和眼压（IOP）进行在体检测，并利用荧光显微镜检测移植眼角膜内皮细胞的CM—DiI标记。数据处理均采用单因素方差分析。结果体外构建的TE—HCE透明度高，具有与活体角膜内皮近似的形态结构，单层细胞密度高达（3602±45）个／mm^2，细胞连接蛋白和膜运输蛋白呈阳性表达：术后跟踪观测结果显示．TE—HCE移植眼的角膜没有出现明显的炎症和免疫排斥反应，角膜维持透明，虽然出现了轻度角膜水肿，但随着时间的推移CCT逐渐下降，术后181d时单层细胞密度为（2796±157）个／mm^2；而mdAM移植眼的角膜则出现了明显的炎症反应，长人了新生血管，角膜持续混浊与水肿，厚度没有明显的下降趋势；各组间IOP的变化无明显差异。CM—DiI荧光检测结果显示TE—HCE移植眼的角膜内皮移植区的细胞均带有CM—DiI标记（来自于移植的TE-HCE）。结论体外构建的高密度TE-HCE具有正常的形态结构，且功能蛋白表达正常．移植后能使猕猴角膜逐渐恢复透明度和厚度，表明TE—HCE在体内可重建出功能正常的角膜内皮，有望用于角膜内皮异常疾病的临床治Objective To construct a tissue-engineered human corneal endothelium with normal morphology, histological property, and evaluate its functions in monkey models. Methods Experimental study. Tissue-engineered HCE （TE-HCE） was constructed by culturing monoclonal human corneal endothelial cells （mcHCE cells） on mdAMs in 20% fetal bovine serum-containing DMEM/Ham＇s Nutrient Mixture F12 （1：1） medium and 5% CO2 at 37 ℃ on a 24-well culture plate. The constructed TE-HCE and mdAM were transplanted into monkey corneas via penetrating keratoplasty with Descemet＇s membrane and endothelium stripped 3 right eyes respectively, left eyes as normal control eyes （6 eyes）. The corneal transparency, endothelial cell density, thickness and intraocular pressure were monitored in vivo. Data were analyzed for statistical significance with one way analysis of variance （ANOVA）. Results The constructed TE-HCE, with an average density of 3 602±45 cells/mm2, mimicked its natural counterpart both in morphology and histological structure. In vivo, corneal transparency was maintained with the density of 2 796±157 cells/mm2, and the corneal thickness gradually decreased after TE-HCE transplanted into monkey eyes, while intense corneal edema arid turbid were found in mdAM-transplanted eyes with their corneal thicknesses increased during the monitoring period. Besides, the cells in transplanted area of corneal endothelium, with CM-DiI label, were all the seeder cells from constructed TE-HCE. Conclusion The constructed TE-HCE has normal histological property and functions well in monkey models. The TE-HCE could be used as a promising HCE equivalent in therapy of corneal endothelium dysfunction and corneal regenerative medicine.

关 键 词：组织工程 内皮 角膜 角膜移植 猕猴属 体外构建 

分 类 号：R779.65[医药卫生—眼科]