【摘要】 目的:验证体外重建组织工程人角膜内皮(TE-HCE)在角膜内皮移植中的作用。方法:以非转染人角膜内皮细胞(HCE细胞)为种子细胞,以去上皮层修饰羊膜为载体支架体外重建的TE-HCE,经CM-DiI标记后对撕除内皮层和后弹力层(DM)的新西兰兔进行了兔穿透性角膜内皮移植。用裂隙灯显微镜观察移植眼角膜的透明度。用荧光显微镜观察种子细胞荧光标记。用茜素红染色和冰冻切片HE染色和扫描电镜观察种子细胞的形态、细胞连接的形成情况、细胞单层的完整性及其与DM结合的紧密程度。用透射电镜方法鉴定种子细胞、DM和角膜的超微结构。结果:TE-HCE可使新西兰兔角膜保持透明39d以上。角膜内皮层移植区的细胞均带有CM-DiI荧光标记。绝大多数种子细胞为六角形,细胞间连接紧密,重建出了完整的角膜内皮层,且内皮层与DM结合紧密。种子细胞重建出了连续的角膜内皮层,种子细胞、DM和角膜的形态结构与正常对照眼的几乎相同。结论:移植后的TE-HCE在种子细胞形态、连续单层状态、细胞连接形成及超微结构上均与对照兔眼的角膜内皮类似,使移植新西兰兔角膜长期保持透明。更多还原

【Abstract】 AIM:To investigate biological functions of in vitro reconstructed tissue-engineered human corneal endothelia(TE-HCE) by animal corneal endothelium transplantation.METHODS:TE-HCEs,reconstructed by using untransfected human corneal endothelialcells(HCE cells,labeled with CM-DiI) as seed cells and modified denuded amniotic membrane(mdAM) as scaffold carriers,were used for penetrating corneal endothelium transplantation in New Zealand white rabbits whose corneal endothelium along with Descemet’s membrane(DM) was ripped off before transplantation.The corneal transparency was monitored with a slit-lamp biomicroscope,and the CM-DiI label of seed cells was checked with a fluorescent microscope.The morphology of seed cells,formation of cell junctions,integrality of endothelial monolayer and its integrated status to DM were investigated by Alizarin red staining,freeze-section’s hematoxylin-eosin(HE) staining and scanning electron microscopy.The ultrastructure of seed cells,DM and corneas were examined by transmission electron microscopy.RESULTS:Slit-lamp biomicroscopic observation of transplanted eyes showed that the TE-HCEs could maintain corneal transparency of the transplanted New Zealand white rabbits for more than 39 days.Fluorescent observations showed that all the seed cells in the transplanted area had positive CM-DiI labels.Alizarin red staining,freeze-section’s HE staining and scanning electron microscopic detections showed that most of seed cells were in hexagonal morphology,integral endothelial monolayer was reconstructed with tight intercellular junctions,and endothelial monolayer integrated tightly to DM,secreted from seed cells.Transmission electron microscopic examination showed that a continuous endothelial monolayer was reconstructed by transplanted TE-HCE,and the ultrastructures of seed cells,DM and corneas were almost the same with those from control eyes.CONCLUSION:The cell morphology,status of continuous monolayer,cell junction and ultrastructure of transplanted TE-HCEs are almost the same with those of rabbit corneal endothelia from control eyes.The TE-HCEs,with similar structures and functions to those of rabbit corneal endothelia,have abilities of maintaining long term cornea transparency of New Zealand white rabbits,and may be used promisingly as HCE equivalents for clinical corneal endothelium transplantation.更多还原