组织工程人角膜内皮的体外重建及其形态结构

In vitro reconstruction of tissue-engineered human corneal endothelium and characterization of its morphology and structure

目的:组织工程人角膜内皮(tissue-engineered human corneal endothelia,TE-HCE)的体外重建及其形态结构。方法:用有限稀释法从HCE细胞系筛选出单克隆细胞(mcHCE细胞),用常规染色体标本制作和核型分类学方法进行核型分析。用羊膜的胰酶倒置消化和细胞外基质蛋白包被方法制备去上皮层修饰羊膜(mdAM)。以核型正常的对数期mcHCE细胞为种子细胞,以平铺于24孔培养板孔底的mdAM为载体支架,用200mL/L胎牛血清-DMEM/F12培养液在37℃,50mL/LCO2培养箱中进行TE-HCE的体外重建。用茜素红染色、冰冻切片HE染色、倒置显微镜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mdAM结合的紧密程度。用透射电镜方法鉴定种子细胞的超微结构以及细胞连接的形成情况。用免疫荧光技术检测种子细胞对不同细胞连接蛋白的表达模式。结果:从非转染HCE细胞系中筛选出了7个核型正常(2n=46)的单克隆细胞株。在启动重建30h后,mcHCE种子细胞在mdAM上形成了完整的细胞单层,细胞密度高达3413/mm2。HCE细胞呈多角形细胞形态,形成了完整的细胞单层,且在细胞-细胞以及细胞-mdAM间形成了多种细胞连接,种子细胞在超微结构上与在体HCE细胞类似,胞质中含有许多线粒体,并具有紧密连接蛋白-1、钙黏蛋白、间隙连接蛋白-43和整联蛋白αv/β5的阳性表达。结论:体外重建的TE-HCE在结构和功能上与在体HCE相似,有望作为HCE的替代物用于临床角膜内皮移植。

AIM： To reconstruct tissue-engineered human corneal endothelia （TE-HCE） in vitro and characterize them in morphology and structure. METHODS： Monoclonal HCE cells （mcHCE cells） were cloned from untransfected HCE cell line by limited dilution, and their karyotypes were analyzed by routine methods of chromosomal preparing and karyosystematics. Modified denuded amniotic membranes （mdAMs） were prepared from amniotic membrane by inverted trypsin denudation and coated with extracellular matrix proteins. TE-HCEs were in vitro reconstructed by using mcHCE cells at logarithmic phase as seed cells and mdAMs tiled on well bottoms of a 24-well culture plate as scaffold carriers, which were cultured in 200mL/L fetal bovine serum （ FBS）-containing DMEM/F12 medium at 37℃ in a 50mL/L CO2 incubator. The morphology of seed cells, formation of cell junctions, integrality of endothelial monolayer and its integrated status to mdAM were investigated by Alizarin red staining, freeze-section ＇s hematoxylin-eosin （HE） staining, inverted microscopy and scanning electron microscopy. The ultrastructure of seed cells on mdAM and formation of cell junctions were examined by transmission electron microscopy. The expression patterns of different cell junction proteins of TE-HCE seeder cells were detected by immunofluorescent techniques. RESULTS： Seven mcHCE cell strains with normal karyotype （2n = 46） were screened out from the untransfected HCE cell line. About 30 hours after reconstruction initiation, mcHCE seeder cells formed an integrated monolayer on mdAM with a cell density as high as 3 413/mm^2. Most of seed cells were in polygonal morphology, integral endothelial monolayer was reconstructed with various cell-cell and cell-mdAM junctions. And the ultrastructure of seed cells was similar to that of HCE cells in vivo, with a lot of mitochondria scattered in cytoplasm. Besides, the seed cells maintained positive expression of cell junction proteins such as zonula occludens protein 1, N-cadherin, connecxin-43 and integrin αv/β5. CONCLSUION. The TE-HCEs, with similar morphology and structure to those of HCE in vivo, were successfully reconstructed, and can be used promisingly as HCE equivalents for clinical corneal endothelium transplantation.