Fan Ting-Jun, Xu Bin, Zhao Jun, Yang Hong-Shou, Wang Rui-Xin, Hu Xiu-Zhong
Key Laboratory for Corneal Tissue Engineering, Ocean University of China, Qingdao 266003, Shandong Province, China.
Int J Ophthalmol. 2011;4(3):228-34. doi: 10.3980/j.issn.2222-3959.2011.03.02. Epub 2011 Jun 18.
To establish an untransfected human corneal epithelial (HCEP) cell line and characterize its biocompatibility with denuded amniotic membrane (dAM).
The torn HCEP pieces were primarily cultured in DMEM/F12 media (pH 7.2) supplemented with 20% fetal bovine serum and other necessary factors, yielding an HCEP cell line which was its growth performance, chromosome morphology, tumorigenicity and expression of marker proteins analyzed. In addition, the biocompatibility of HCEP cells with dAM was evaluated through histological and immunocytochemistry analyses and with light, electron and slit-lamp microscopies.
HCEP cells proliferated to confluence in 3 weeks, which have been subcultured to passage 160. A continuous untransfected HCEP cell line, designated as utHCEPC01, was established with a population doubling time of 45.42 hours as was determined at passage 100. The cells retained HCEP cell properties as were approved by chromosomal morphology and the expression of keratin 3. They, with no tumorigenicity, formed a multilayer epithelium-like structure on dAMs through proliferation and differentiation during air-liquid interface culture, maintained expression of marker proteins including keratin 3 and integrin β1 and attached tightly to dAMs. The reconstructed HCEP was highly transparent and morphologically and structurally similar to the original.
An untransfected and non-tumorigenic HCEP cell line was established in this study. The cells maintained expression of marker proteins. The cell line was biocompatible with dAM. It holds the potential of being used for in vitro reconstruction of tissue-engineered HCEP, promising for the treatment of diseases caused by corneal epithelial disorders.
建立未转染的人角膜上皮（HCEP）细胞系，并表征其与脱细胞羊膜（dAM）的生物相容性。
将撕裂的HCEP碎片在补充有20%胎牛血清和其他必要因子的DMEM/F12培养基（pH 7.2）中进行原代培养，获得HCEP细胞系，并对其生长性能、染色体形态、致瘤性和标记蛋白表达进行分析。此外，通过组织学和免疫细胞化学分析以及光镜、电镜和裂隙灯显微镜评估HCEP细胞与dAM的生物相容性。
HCEP细胞在3周内增殖至汇合，已传代至第160代。建立了一个连续的未转染HCEP细胞系，命名为utHCEPC01，在第100代时测定其群体倍增时间为45.42小时。细胞保留了HCEP细胞特性，这通过染色体形态和角蛋白3的表达得以证实。它们无致瘤性，在气液界面培养期间通过增殖和分化在dAM上形成多层上皮样结构，维持包括角蛋白3和整合素β1在内的标记蛋白表达，并紧密附着于dAM。重建的HCEP高度透明，在形态和结构上与原始组织相似。
本研究建立了未转染且无致瘤性的HCEP细胞系。细胞维持标记蛋白表达。该细胞系与dAM具有生物相容性。它具有用于组织工程化HCEP体外重建的潜力，有望用于治疗角膜上皮疾病引起的病症。
