Hongo Akane, Okumura Naoki, Nakahara Makiko, Kay EunDuck P, Koizumi Noriko
Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
Invest Ophthalmol Vis Sci. 2017 Jul 1;58(9):3325-3334. doi: 10.1167/iovs.16-21170.
We have begun a clinical trial of a cell-based therapy for corneal endothelial dysfunction in Japan. The purpose of this study was to investigate the usefulness of a p38 MAPK inhibitor for prevention cellular senescence in cultivated human corneal endothelial cells (HCECs).
HCECs of 10 donor corneas were divided and cultured with or without SB203580 (a p38 MAPK inhibitor). Cell density and morphology were evaluated by phase-contrast microscopy. Expression of function-related proteins was examined by immunofluorescent staining. Cellular senescence was evaluated by SA-β-gal staining and Western blotting for p16 and p21. Senescence-associated factors were evaluated by membrane blotting array, quantitative PCR, and ELISA.
Phase-contrast microscopy showed a significantly higher cell density for HCECs cultured with SB203580 than without SB203580 (2623 ± 657 cells/mm2 and 1752 ± 628 cells/mm2, respectively). The HCECs cultured with SB203580 maintained a hexagonal morphology and expressed ZO-1, N-cadherin, and Na+/K+-ATPase in the plasma membrane, whereas the control HCECs showed an altered staining pattern for these marker proteins. HCECs cultured without SB203580 showed high positive SA-β-gal staining, a low nuclear/cytoplasm ratio, and expression of p16 and p21. IL-6, IL-8, CCL2, and CXCL1 were observed at high levels in low cell density HCECs cultured without SB203580.
Activation of p38 MAPK signaling due to culture stress might be a causative factor that induces cellular senescence; therefore, the use of p38 MAPK inhibitor to counteract senescence may achieve sufficient numbers of HCECs for tissue engineering therapy for corneal endothelial dysfunction.
我们在日本已启动一项针对角膜内皮功能障碍的细胞疗法临床试验。本研究的目的是调查p38丝裂原活化蛋白激酶（MAPK）抑制剂在预防培养的人角膜内皮细胞（HCEC）细胞衰老方面的有效性。
将10个供体角膜的HCEC进行分组，分别在有或无SB203580（一种p38 MAPK抑制剂）的条件下培养。通过相差显微镜评估细胞密度和形态。通过免疫荧光染色检测功能相关蛋白的表达。通过衰老相关β-半乳糖苷酶（SA-β-gal）染色以及针对p16和p21的蛋白质印迹法评估细胞衰老。通过膜印迹阵列、定量聚合酶链反应（PCR）和酶联免疫吸附测定（ELISA）评估衰老相关因子。
相差显微镜显示，与未使用SB203580培养的HCEC相比，使用SB203580培养的HCEC细胞密度显著更高（分别为2623±657个细胞/mm²和1752±628个细胞/mm²）。使用SB203580培养的HCEC保持六边形形态，并在质膜中表达紧密连接蛋白1（ZO-1）、N-钙黏蛋白和钠钾ATP酶，而对照HCEC显示这些标记蛋白的染色模式发生改变。未使用SB203580培养的HCEC显示SA-β-gal染色呈高阳性、核/质比低以及p16和p21表达。在未使用SB203580培养的低细胞密度HCEC中观察到白细胞介素-6（IL-6）、白细胞介素-8（IL-8）、趋化因子配体2（CCL2）和趋化因子配体1（CXCL1）水平较高。
培养应激导致的p38 MAPK信号激活可能是诱导细胞衰老的一个致病因素；因此，使用p38 MAPK抑制剂对抗衰老可能为角膜内皮功能障碍的组织工程治疗获得足够数量的HCEC。
