Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago IL, USA.
Sci Rep. 2017 Jan 5;7:40308. doi: 10.1038/srep40308.
Rapamycin has previously been shown to have anti-aging effects in cells and organisms. These studies were undertaken to investigate the effects of rapamycin on primary human corneal epithelial cells in vitro. Cell growth and viability were evaluated by bright field microscopy. Cell proliferation and cycle were evaluated by flow cytometry. The expression of differentiation markers was evaluated by quantitative PCR and Western blot. Senescence was evaluated by senescence-associated β-Galactosidase staining and by Western blot analysis of p16. Apoptosis was evaluated by a TUNEL assay. The results demonstrated that primary HCEC treated with rapamycin had lower proliferation but considerably longer survival in vitro. Rapamycin-treated cells maintained a higher capacity to proliferate after removal of rapamycin and expressed more keratin 14, N-Cadherin, DeltaNp63 and ABCG2, and less keratin 12, consistent with their less differentiated state. Rapamycin treated cells demonstrated less senescence by X-β-Gal SA staining and by lower expression of p16. Apoptosis was also lower in the rapamycin treated cells. These results indicate that rapamycin treatment of HCEC prevents the loss of corneal epithelial stem/progenitor cells to replicative senescence and apoptosis. Rapamycin may be a useful additive for ex vivo expansion of corneal epithelial cells.
雷帕霉素先前已被证明在细胞和生物体内具有抗衰老作用。这些研究旨在探讨雷帕霉素对体外原代人角膜上皮细胞的影响。通过明场显微镜评估细胞生长和活力。通过流式细胞术评估细胞增殖和周期。通过定量 PCR 和 Western blot 评估分化标志物的表达。通过衰老相关β-半乳糖苷酶染色和 p16 的 Western blot 分析评估衰老。通过 TUNEL 测定评估细胞凋亡。结果表明，用雷帕霉素处理的原代 HCEC 体外增殖率较低，但存活时间显著延长。雷帕霉素处理的细胞在去除雷帕霉素后仍保持较高的增殖能力，表达更多的角蛋白 14、N-钙黏蛋白、DeltaNp63 和 ABCG2，而角蛋白 12 表达减少，表明其分化程度较低。X-β-半乳糖苷酶 SA 染色和 p16 表达降低表明雷帕霉素处理的细胞衰老减少。雷帕霉素处理的细胞凋亡也较低。这些结果表明，雷帕霉素处理可防止角膜上皮干细胞/祖细胞因复制性衰老和凋亡而丧失。雷帕霉素可能是角膜上皮细胞体外扩增的有用添加剂。
