Rho-associated protein kinase inhibitor, Y-27632, significantly enhances cell adhesion and induces a delay in G1 to S phase transition in rabbit corneal endothelial cells
Human corneal endothelial cells are a non-proliferative cell type. As a result of the increase in corneal endothelium disease, increasing numbers of studies have been conducted in order to promote corneal endothelial cell proliferation. The aim of the present study was to investigate the proliferative effects of Rhoassociated protein kinase inhibitor, Y27632, on rabbit corneal endothelial cells (rCECs). Y27632 (1, 10 or 30 µM) was added at two different time points to two groups of rCECs. The first group received Y27632 when rCECs were initially plated, and the second following 72 h of cell growth. Cell morphology and cell adhesion ratios were subsequently observed using light microscopy. A cell counting kit was used to measure the number of viable cells that adhered to culture plates. Cell cycle transitions and levels of Annexin Vpositive apoptotic cells were detected using flow cytometry. Cells treated with 1 µM Y27632 and 10 µM Y27632 retained their cell shape. At a concentration of 30 µM Y27632, the cell shape became irregular. Cell adhesion ratios, in 1 µM Y27632 (36.84%), 10 µM Y27632 (84.21%) and 30 µM Y27632 (84.21%) were higher than the adhesion ratio in the control group (P<0.01). The optical densities of rCECs treated with 10 µM or 30 µM Y27632 following 72 h of cell growth was less than that of the control cells (P<0.01), but higher than that of cells which received Y27632 at the time of plating (P<0.01). Flow cytometry results also demonstrated that there was a delay in G1 to S phase cell cycle progression in rCECs following administration of 10 µM Y27632 (P<0.01). Cell apoptosis was inhibited when 10 µM Y27632 was added, at the time of cell plating, as well as when added following 72 h of cell growth (P<0.01). At a concentration of 10 µM Y27632, there was an improvement in cell adhesion and an inhibition of the cell cycle in rabbit corneal endothelial cells. In conclusion, Y27632 has different effects on rCECs when administered at varying concentrations and at particular stages of cell growth.
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Document Type: Research Article
Affiliations: Department of Ophthalmology, Peking University Third Hospital, Haidian, Beijing 100191, P.R. China
Publication date: August 1, 2015
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