Robertson Danielle M, Li Li, Fisher Stephen, Pearce Virginia P, Shay Jerry W, Wright Woodring E, Cavanagh H Dwight, Jester James V
Department of Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.
Invest Ophthalmol Vis Sci. 2005 Feb;46(2):470-8. doi: 10.1167/iovs.04-0528.
To develop and characterize a telomerase-immortalized human corneal epithelial cell line (hTCEpi) to serve as an in vitro model for studying the molecular mechanisms involved in regulating human corneal epithelial cell differentiation.
Primary cultures of human corneal epithelial cells were infected with a retroviral vector encoding human telomerase reverse transcriptase (hTERT). Infected hTCEpi cells were selected, cloned, and characterized to identify telomerase activity, proliferative capacity, karyotype, and differentiative potential in routine culture and under consecutive submerged and air-lifted conditions. Cells were evaluated to measure cell cycle kinetics (anti-Ki-67, anti-p16), stratification (phalloidin and anti-ZO-1), and differentiation (anti-K3, anti-BCL-2 and TUNEL labeling).
hTCEpi cells exhibited telomerase activity, a normal karyotype and cell cycle kinetics at greater than 240 population doublings, and loss of p16 after passage 10. Air-lifting produced a well stratified epithelium (five to seven cell layers) with apical ZO-1-stained tight junctions. Submersed culture demonstrated increasing expression of stratification markers (K5/K14) with K3-corneal keratin marker expression in long-term, air-lifted culture. Anti-BCL-2 staining showed both nuclear and cytoplasmic localization with loss of nuclear BCL-2 expression in TUNEL-labeled surface epithelial cells.
hTCEpi cells stratify, differentiate, and desquamate similar to normal human corneal epithelium. Further study of the hTCEpi cell line may be valuable in studying the molecular mechanisms regulating corneal epithelial cell differentiation and desquamation.
开发并鉴定一种端粒酶永生化的人角膜上皮细胞系（hTCEpi），用作体外模型以研究参与调控人角膜上皮细胞分化的分子机制。
用人角膜上皮细胞原代培养物感染编码人端粒酶逆转录酶（hTERT）的逆转录病毒载体。对感染的hTCEpi细胞进行筛选、克隆和鉴定，以确定其在常规培养以及连续浸没和空气提升条件下的端粒酶活性、增殖能力、核型和分化潜能。通过测量细胞周期动力学（抗Ki-67、抗p16）、分层（鬼笔环肽和抗ZO-1）以及分化（抗K3、抗BCL-2和TUNEL标记）对细胞进行评估。
hTCEpi细胞在超过240次群体倍增时表现出端粒酶活性、正常核型和细胞周期动力学，传代10次后p16表达缺失。空气提升培养产生了分层良好的上皮（五到七层细胞），顶端有ZO-1染色的紧密连接。浸没培养显示分层标记物（K5/K14）表达增加，长期空气提升培养中有角膜角蛋白标记物K3表达。抗BCL-2染色显示细胞核和细胞质均有定位，TUNEL标记的表面上皮细胞中细胞核BCL-2表达缺失。
hTCEpi细胞的分层、分化和脱屑与正常人角膜上皮相似。对hTCEpi细胞系的进一步研究可能对研究调控角膜上皮细胞分化和脱屑的分子机制具有重要价值。
Zotero 插件