Fan Tingjun, Wang Dansheng, Zhao Jun, Wang Jing, Fu Yongfeng, Guo Ruichao
Research Institute of Corneal Tissue Engineering, Ocean University of China, Shandong, China.
Mol Vis. 2009 May 29;15:1070-8.
To establish and characterize a novel untransfected corneal endothelial cell line from New Zealand white rabbits (NRCE cell line) for studies on corneal endothelial cells.
Primary culture was initiated with a pure population of NRCE cells from corneal endothelia by successive detachment and reattachment procedure of different durations, and cultured in 20% fetal bovine serum-containing DMEM/F12 media with several supplements. The cell line was characterized by chromosome analysis, fluorescence immunoassay and reverse transcription PCR. The tumorigenic potency of the cell line was examined by subcutaneous inoculation to nude mice. The biocompatibility of the cell line to denuded amnions was examined with routine microscopic and electron microscopic techniques.
NRCE cells in primary culture proliferated to confluency in 25 days and has been subcultured to passage 227 to date. The novel NRCE cell line, with a steady growing rate in 20% bovine calf serum (BCS)-containing DMEM/F12 medium and a population doubling time of 40.32 h at passage 191, has been established. NRCE cells exhibited chromosomal aneuploidy but their modal chromosome number was still 44. The results of gene expression patterns of marker proteins and membrane transport proteins, combined with immunofluorescent localization patterns of cell junction proteins, indicated that NRCE cells retained normal corneal endothelial characteristics and normal expression pattern of functional proteins. Furthermore, these cells, without any tumorigenic potency, had excellent biocompatibility to denuded amnions in 20% BCS-containing DMEM/F12 medium, and formed confluent cell sheets attached tightly to denuded amnions.
These results suggest that a novel untransfected NRCE cell line established here maintains normal corneal endothelial characteristics and potencies to form normal cell junctions and perform normal functions of transmembrane transport.
建立并鉴定一种来自新西兰白兔的新型未转染角膜内皮细胞系（NRCE细胞系），用于角膜内皮细胞研究。
通过不同时长的连续分离和重新贴壁程序，从角膜内皮获取纯的NRCE细胞群体进行原代培养，并在含有多种添加剂的含20%胎牛血清的DMEM/F12培养基中培养。通过染色体分析、荧光免疫测定和逆转录PCR对该细胞系进行鉴定。通过皮下接种裸鼠检测该细胞系的致瘤潜能。用常规显微镜和电子显微镜技术检测该细胞系对去上皮羊膜的生物相容性。
原代培养的NRCE细胞在25天内增殖至汇合，迄今为止已传代至227代。已建立新型NRCE细胞系，其在含20%小牛血清（BCS）的DMEM/F12培养基中生长速率稳定，在第191代时群体倍增时间为40.32小时。NRCE细胞表现出染色体非整倍性，但其众数染色体数仍为44。标记蛋白和膜转运蛋白的基因表达模式结果，结合细胞连接蛋白的免疫荧光定位模式，表明NRCE细胞保留了正常角膜内皮特征和功能蛋白的正常表达模式。此外，这些细胞无任何致瘤潜能，在含20% BCS的DMEM/F12培养基中对去上皮羊膜具有优异的生物相容性，并形成紧密附着于去上皮羊膜的汇合细胞片。
这些结果表明，在此建立的新型未转染NRCE细胞系维持了正常角膜内皮特征以及形成正常细胞连接和执行跨膜转运正常功能的能力。
