Department of Ophthalmology, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9057, USA.
Curr Eye Res. 2011 Aug;36(8):706-12. doi: 10.3109/02713683.2011.582662.
Telomerase-immortalized human corneal epithelial cells have been reported to stratify and differentiate in vitro similar to native tissue. The purpose of this study was to assess the ability of a telomerase-immortalized human corneal epithelial cell line to generate a full thickness epithelium in vivo in athymic mice.
Telomerized corneal epithelial cells were transduced with a retroviral vector encoding the herpes simplex thymidine kinase gene. Efficacy of the thymidine kinase suicide gene was confirmed using a live/dead assay. The epithelium was mechanically removed from athymic nude mice and remaining cells were treated with mitomycin C to prevent re-epithelialization. Telomerized corneal epithelial cells were seeded onto the denuded cornea and allowed to adhere for 4 and 24 hours. Cellular attachment was assessed using a fluorescent cell tracker. Stratification and differentiation were assessed after 7 days using phalloidin and a mouse monoclonal antibody to K3.
Telomerized corneal epithelial cells were visualized across the denuded stromal surface at 4 and 24 hours, with multi-layering evident at the latter time point. No epithelium was present in the non-treated eye. After 7 days post-transplantation cells stratified into a multilayered epithelium, with positive K3 expression in basal and suprabasal cells. Treatment with ganciclovir induced significant loss of viability in vitro.
The findings in this pilot study demonstrate that telomerized corneal epithelial cells possess the capacity to reconstitute a stratified corneal epithelium in vivo. The introduction of thymidine kinase allowed for the successful induction of cell death in proliferating cells in vitro. Collectively, these data suggest that a telomerase-immortalized corneal epithelial cell line transduced with thymidine kinase represents a potential model for studying differentiation and epithelial-niche interactions in vivo with potential applications in tissue engineering.
已报道端粒酶永生化的人角膜上皮细胞在体外具有分层和分化的能力，类似于原组织。本研究的目的是评估端粒酶永生化人角膜上皮细胞系在裸鼠体内产生全层上皮的能力。
用逆转录病毒载体转导端粒化角膜上皮细胞，该载体编码单纯疱疹胸苷激酶基因。使用死活检测法证实胸苷激酶自杀基因的效力。从无胸腺裸鼠中机械去除上皮，并对剩余细胞用丝裂霉素 C 处理以防止再上皮化。将端粒化角膜上皮细胞接种到去上皮角膜上，并允许其附着 4 和 24 小时。使用荧光细胞追踪剂评估细胞附着。7 天后，使用鬼笔环肽和抗 K3 的小鼠单克隆抗体评估分层和分化。
在 4 和 24 小时时，端粒化角膜上皮细胞可见于裸露的基质表面上，后者时间点显示出多层化。未经处理的眼睛中没有上皮。移植后 7 天，细胞分层为多层上皮，基底和超基底细胞中 K3 表达阳性。体外给予更昔洛韦诱导细胞活力显著丧失。
本初步研究结果表明，端粒化角膜上皮细胞具有在体内重建分层角膜上皮的能力。胸苷激酶的引入允许成功诱导体外增殖细胞的细胞死亡。总的来说，这些数据表明，转导了胸苷激酶的端粒酶永生化角膜上皮细胞系代表了研究体内分化和上皮-基质相互作用的潜在模型，具有在组织工程中潜在应用。
