Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology; Department of Ophthalmology, Ninth People's Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, China.
Cornea. 2021 Feb 1;40(2):203-214. doi: 10.1097/ICO.0000000000002560.
To investigate the proliferation of umbilical cord blood-derived endothelial progenitor cells (UCB EPCs) and the differentiation efficiency toward corneal endothelial cell (CEC)-like cells induced by rho-associated protein kinase (ROCK) inhibitor Y-27632 and to determine the most effective strategy for repairing corneal endothelium injuries in rabbits.
UCB EPCs were cultured in Endothelial Cell Growth Medium-2 (EGM-2) media or conditioned media (CM) from human CECs, with and without the addition of Y-27632. Bromo-deoxyuridine (BrdU) immunocytochemistry and cell counting kit-8 assays were used to examine the proliferation of the cells. Real-time polymerase chain reaction, western blot, and immunocytochemistry were used to detect the CEC markers. Nd:YAG laser was used to establish an appropriate endothelium injury model based on rabbit corneas. The following intracameral injections were then performed to repair the model: 100 μL Opti-MEM I reduced serum medium (model group), 2 × 105 UCB EPCs diluted in 100 μL Opti-MEM I reduced serum medium (EPC group), 100 μM Y-27632 diluted in 100 μL Opti-MEM I reduced serum medium (Y-27632 group), and 2 × 105 UCB EPCs supplemented with 100 μM Y-27632 (final volume 100 μL, EPC/Y-27632 group). The follow-up tests focused on corneal transparency, central corneal thickness, intraocular pressure, and in vivo confocal microscopy, which were performed to evaluate the healing of the wounds.
Culturing UCB EPCs in CM supplemented with 10 μM Y-27632 resulted in higher proliferation rates compared with EGM-2 media and CM. There were significantly improved protein levels of Zona Occludens 1, N-cadherin, Na+-K+-ATPase α1, Na+-K+-ATPase β1, and Pax6 and improved mRNA levels of collagen type IV and VIII and AQP1. The combined intracameral injection of Y-27632 and UCB EPCs accelerated the recovery of corneal transparency, regression of corneal edema, and healing of the corneal endothelium compared with the injections of Y-27632 and UCB EPCs on their own.
Y-27632 not only promotes the proliferation of UCB EPCs but also contributes to differentiation of UCB EPCs toward CECs in the presence of CM. The intracameral injection of Y-27632 itself promotes the healing of corneal endothelium wounds. On this basis, supplementing UCB EPCs with Y-27632 accelerates the healing of corneal endothelium wounds.
研究 rho 相关蛋白激酶（ROCK）抑制剂 Y-27632 对脐带血来源的内皮祖细胞（UCB EPC）增殖的影响，以及其向角膜内皮样细胞（CEC）分化的效率，并确定修复兔角膜内皮损伤的最有效策略。
将 UCB EPC 分别在含有人 CEC 条件培养基（CM）的 EGM-2 培养基和无 CM 的 EGM-2 培养基中培养，并加入或不加入 Y-27632。BrdU 免疫细胞化学和细胞计数试剂盒-8 检测细胞增殖。实时聚合酶链反应、western blot 和免疫细胞化学检测 CEC 标志物。Nd：YAG 激光在兔角膜上建立合适的内皮损伤模型。然后进行以下眼内注射修复模型：100μL Opti-MEM I 低血清培养基（模型组）、100μL Opti-MEM I 低血清培养基中稀释的 2×105 UCB EPC（EPC 组）、100μM Y-27632 稀释于 100μL Opti-MEM I 低血清培养基（Y-27632 组）和 100μL Opti-MEM I 低血清培养基中补充 100μM Y-27632 的 2×105 UCB EPC（终体积 100μL，EPC/Y-27632 组）。后续的测试集中在角膜透明度、中央角膜厚度、眼内压和体内共聚焦显微镜上，以评估伤口的愈合情况。
与 EGM-2 培养基和 CM 相比，在含 10μM Y-27632 的 CM 中培养 UCB EPC 可导致更高的增殖率。ZO-1、N-钙黏蛋白、Na+-K+-ATPaseα1、Na+-K+-ATPaseβ1 和 Pax6 的蛋白水平显著改善，胶原 IV 和 VIII 型和 AQP1 的 mRNA 水平也显著改善。与单独注射 Y-27632 和 UCB EPC 相比，联合注射 Y-27632 和 UCB EPC 可加速角膜透明度的恢复、角膜水肿的消退和角膜内皮的愈合。
Y-27632 不仅促进 UCB EPC 的增殖，而且有助于在 CM 存在的情况下促进 UCB EPC 向 CEC 分化。眼内注射 Y-27632 本身可促进角膜内皮伤口的愈合。在此基础上，用 Y-27632 补充 UCB EPC 可加速角膜内皮伤口的愈合。
