Enhancement on Primate Corneal Endothelial Cell Survival In Vitro by a ROCK Inhibitor
Enhancement on Primate Corneal Endothelial Cell Survival In Vitro by a ROCK Inhibitor
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DOI：
10.1167/iovs.08-2634
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发表时间：
2009-08-01
影响因子：
4.4
通讯作者：
Kinoshita, Shigeru
中科院分区：
文献类型：
--
作者：
Okumura, Naoki;Ueno, Morio;Kinoshita, Shigeru
目的.培养的角膜内皮细胞（CEC）移植治疗角膜内皮功能障碍的患者近年来受到关注。然而，用于人（H）CEC的有效培养技术尚未适当建立。本研究旨在探讨Rho激酶（ROCK）抑制剂Y-27632在促进食蟹猴CEC培养中的适用性。在含有10 μ M Y-27632的培养基中培养食蟹猴的MCEC。通过发光细胞活力测定监测粘附于培养板的活细胞的数量，并通过甲苯胺蓝染色检测集落生长。使用流式细胞术和BrdU标记的免疫细胞化学测定通过Ki 67表达检测增殖细胞。通过流式细胞术分析膜联蛋白V阳性凋亡细胞。培养24小时后，通过添加Y-27632增加了活的培养MCEC的数量。在第10天，存在Y-27632的培养物的殖民地面积高于不存在Y-27632的培养物。在Y-27632处理的培养物中，Ki 67阳性细胞的数量在24和48小时显著增加，并且增殖的BrdU阳性细胞的数量在48小时增加。Annexin V阳性凋亡细胞的数量在24小时减少。特异性ROCK抑制剂Y-27632抑制Rho/ROCK信号通路可促进MCECs的粘附，抑制凋亡，增加增殖细胞数。这些结果表明，ROCK抑制剂可以作为一种新的工具，培养HCECs移植。（Invest Ophthalmol维斯科学。2009;50：3680-3687）DOI：10.1167/iovs.082634
PURPOSE. The transplantation of cultivated corneal endothelial cells (CECs) has gained attention recently for the treatment of patients with corneal endothelial dysfunction. However, an efficient culturing technique for human (H) CECs has yet to be properly established. The present study was conducted to investigate the applicability of the Rho kinase (ROCK) inhibitor Y-27632 in promoting cultivation of cynomolgus monkey (M) CECs.METHODS. MCECs of cynomolgus monkeys were cultured in a medium containing 10 mu M Y-27632. The number of viable cells adherent to culture plates were monitored by a luminescent cell-viability assay and colony growth was detected by toluidine blue staining. Proliferating cells were detected by Ki67 expression using flow cytometry and a BrdU-labeling assay for immunocytochemistry. Annexin V-positive apoptotic cells were analyzed by flow cytometry.RESULTS. The number of viable cultivated MCECs was enhanced by Y-27632 addition after 24 hours in culture. The colony area of the culture in the presence of Y-27632 was higher than in the absence of Y-27632 on day 10. In Y-27632-treated cultures, the number of Ki67-positive cells was significantly increased at 24 and 48 hours, and the number of proliferating BrdU-positive cells was increased at 48 hours. The number of Annexin V-positive apoptotic cells was decreased at 24 hours.CONCLUSIONS. The inhibition of Rho/ROCK signaling by specific ROCK inhibitor Y-27632 promoted the adhesion of MCECs, inhibited apoptosis, and increased the number of proliferating cells. These results suggest that the ROCK inhibitor may serve as a new tool for cultivating HCECs for transplantation. (Invest Ophthalmol Vis Sci. 2009;50:3680-3687) DOI: 10.1167/iovs.082634