Shenzhen Eye Hospital, Shenzhen, Guangdong 518040, China; Shenzhen R&D Center of State Key Laboratory of Virology, Wuhan University Shenzhen Institute, Shenzhen, Guangdong 518057, China; State Key Laboratory of Virology, Institute of Medical Virology, Wuhan University School of Basic Medical Sciences, Wuhan, Hubei 430071, China.
Shenzhen Eye Hospital, Shenzhen, Guangdong 518040, China; Visual Optics Institute, Health Science Center, Shenzhen University, Shenzhen, Guangdong 518060, China.
Biochem Biophys Res Commun. 2018 May 23;499(4):735-742. doi: 10.1016/j.bbrc.2018.03.168. Epub 2018 Apr 10.
In this study, we established human limbal epithelial cells (LECs) from normal limbal tissues by using Conditional Reprogramming (CR) technology (refer to CR-LEC cells in this study). We have successfully established CR-LEC cell strains from three human donors (3 out of 3), and normal rabbits (2 out of 2) and pig (1 out of 1) as well. CR-LEC cells sustained a continuous and stable proliferation status with a normal karyotype, normal response to DNA damage, well-defined structured spheres in matrigel 3D culture. Responses of CR-LEC cells to IFN α2b, Ganciclovir and 5-Fluorouracil were different, suggesting that these drugs had different toxicities to these cells as expected. More important, there was no significant difference of responses to drugs between early and late passages of CR-LEC cells (p>0.05), indicating CR-LEC cells can serve a stable normal human cell model for toxicity assessment. Toxicity tests with monolayer cultures of CR-LEC cells were measured by staining the F-actin and Dsg-1 expression. Toxicity of three drugs at LD50 concentration resulted in a gradually increased destruction of monolayer, which is, in accordance with the irritation grade of three drugs on human cornea epithelium. Therefore, CR-LEC cells provide a novel and reliable in vitro physiological cell model for corneal toxicity assessment.
在这项研究中，我们使用条件重编程（CR）技术（在本研究中称为 CR-LEC 细胞）从正常角膜组织中建立了人角膜缘上皮细胞（LEC）。我们已经成功地从三个人类供体（3 个中有 3 个）和正常兔子（2 个中有 2 个）以及猪（1 个中有 1 个）建立了 CR-LEC 细胞株。CR-LEC 细胞保持持续稳定的增殖状态，具有正常的核型、对 DNA 损伤的正常反应以及在基质胶 3D 培养中形成规则的球体结构。CR-LEC 细胞对 IFNα2b、更昔洛韦和 5-氟尿嘧啶的反应不同，表明这些药物对这些细胞的毒性预期不同。更重要的是，CR-LEC 细胞早期和晚期传代之间对药物的反应没有显著差异（p>0.05），表明 CR-LEC 细胞可以作为一种稳定的正常人类细胞模型用于毒性评估。通过染色 F-肌动蛋白和 Dsg-1 表达来测量 CR-LEC 细胞单层培养物的毒性试验。LD50 浓度下三种药物的毒性导致单层逐渐破坏，这与三种药物对人角膜上皮的刺激程度一致。因此，CR-LEC 细胞为角膜毒性评估提供了一种新颖可靠的体外生理细胞模型。
