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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。
In order to reconstruct the tissue-engineered corneal endothelium (TE-HCE) that can be used for corneal transplantation in vitro, a monoclonal cell line (mcHCE) was screened from established non-transfected human corneal endothelial cells (HCE) In vitro reconstitution of TE-HCE was studied for seed cells. The mcHCE cell line was screened from the non-transfected HCE cell line by limiting dilution. The morphological structure, chromosome analysis and the detection of cell junction protein and membrane transport protein showed that the mcHCE2401 monoclonal cell line had a normal and stable morphology, structure And double chromosome karyotypes, and express both connexin and membrane transport proteins, with the desirable characteristics of TE-HCE seed cells. Using mcHCE2401 cells as seed cells, TE-HCE was reconstructed in vitro using mdAM as carrier. The morphological structure of mcHCE2401 seed cells showed that the mcHCE2401 seed cells formed a continuous and intact cell monolayer (3 602.22 ± 45.22) cells / mm2 (equivalent to the cell density of HCE in children aged 0-3 years). The reconstructed single The morphology of corneal endothelium is highly similar to that of in vivo HCE. Therefore, we successfully established a monoclonal cell line with morphological structure, karyotype and normal expression of functional protein. The mcHCE2401 cells and mdAM were successfully used to reconstruct the morphological structure and the most similar “young” Of TE-HCE is expected to be used as an equivalent substitute for donated corneal endothelium for the clinical treatment of abnormal corneal endothelium.