人角膜内皮细胞株的建立与组织工程人角膜内皮的体外重建  

Establishment of a human corneal endothelial cell strain and in vitro reconstruction of a tissue-engineered human corneal endothelium

作　　者：樊廷俊[1] 马西亚[1] 赵君[1] 胡修忠[1] 

机构地区：[1]中国海洋大学角膜组织工程重点实验室,山东青岛266003

出　　处：《山东大学学报（理学版）》2011年第10期142-151,共10页Journal of Shandong University(Natural Science)

基　　金：国家高技术研究发展计划(863计划)资助项目(2006AA02A132)

摘　　要：为了体外重建出可用于角膜移植的组织工程人角膜内皮(TE-HCE),本文从业已建立的非转染人角膜内皮(HCE)细胞系中筛选出单克隆细胞株(mcHCE),并以其为种子细胞对TE-HCE的体外重建进行了研究。经有限稀释法从非转染HCE细胞系筛选出了mcHCE细胞株,形态结构、染色体分析以及细胞连接蛋白和膜运输蛋白的检测结果显示,mcHCE2401单克隆细胞株细胞具有正常而稳定的形态、结构和二倍染色体核型,并能表达细胞连接蛋白和膜运输蛋白,具有TE-HCE种子细胞的理想特征。以mcHCE2401细胞为种子细胞、以去上皮层修饰羊膜(mdAM)为载体支架体外重建出了TE-HCE,形态结构鉴定结果显示,多角形mcHCE2401种子细胞在mdAM上形成了连续、完整的细胞单层,在细胞之间以及细胞与mdAM之间均形成了广泛的细胞连接,单层细胞密度高达(3 602.22±45.22)个/mm2(相当于0～3岁孩童HCE的细胞密度),所重建单层角膜内皮的形态结构与在体HCE高度近似。可见,本文成功建立了形态结构、核型以及功能蛋白表达正常的单克隆细胞株,并利用mcHCE2401细胞和mdAM在体外成功重建出了形态结构与与在体HCE高度近似的最"年轻"的TE-HCE,有望作为捐献角膜内皮的等效替代物用于角膜内皮异常疾病的临床治疗。To reconstruct tissue-engineered human corneal endothelium （TE-HCE） suitable for corneal transplantation, this study was intended to screen out monoclonal cell strains from an untransfected human corneal endothelial （HCE） cell line, and reconstruct TE-HCE in vitro by using these cells as seeder cells. Monoclonal cell strains were screened out from the HCE cell line by limit dilution. Results of morphology, structure and chromosome analysis, combined with the results of expression of cell-junction proteins and membrane transport proteins, suggested that the cells from mcHCE2401 monoclonal cell strains had steady and normal morphology, structure, karyotype, and positive expression of cell junction proteins and membrane transport proteins as well. All these imply that mcHCE2401 cells have ideal characteristics of seeder cells for TE-HCE reconstruction. By using mcHCE2401 cells as seeder cells and modified denuded amniotic membrane （mdAM） as scaffold carrier, TE-HCE was reconstructed. Results of morphology and structural examination showed that polygonal mcHCE2401 cells formed a continuous and intact monolayer on mdAM with exten- sive cell-cell and cell-mdAM cell junctions. The average cell density of the monolayer was as high as 3 602. 22 ±45.22 cell/mm^2 （equivalent to HCE cell density of a 0 -3 years old baby）. All these indicate that the reconstructed TE-HCE had very similar morphology and structure to those of HCE in situ. In conclusion, a monoclonal cell strain with normal morphology, structure, karyotype and cell-junction and membrane transport protein expression has been established, and ＂youngest＂ TE-HCE with similar morphology and structure to those of HCE in situ has been successfully reconstructed in vitro and provides a promising equivalent of donated HCE for clinical treatment of diseases caused by corneal endo- thelial disorders.

关 键 词：人角膜内皮细胞 单克隆细胞株 去上皮层修饰羊膜 组织工程人角膜内皮 重建 

分 类 号：R318[医药卫生—生物医学工程] R276.7[医药卫生—基础医学]