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Limbal stem cells (LSCs) play an important role on the stability of corneal epithelium and corneal transparency. Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor can promote cell proliferation and reduce apoptosis, such as human embryonic stem cells and karatin epithelial cells.
This study was to investigate the improving effect of Y-27632, a ROCK inhibitor, on the activity of rabbit LSCs in corneal preservation medium.
Corneal preservation solution was prepared by adding 12. 5% chondroitin sulfate, 10. 0% low molecular dextran, 20. 0 mg/L dexamethasone, 100 mg/L tobramycin sulfate, 9. 5 g/L Hepes and 0. 375 mg/L L-glutamine in MEM. The corneas of New Zealand white rabbits were collected and preserved in the corneal preservation solution with or without Y-27632 for 4, 7, 14 days, and the density and morphology of corneal endothelial cells were examined by using 0. 25% trypan blue staining and 0. 2% alizarin red staining. Isolated corneal epithelial cells were seeded on 3T3 feeder layer and cultured for 7-10 days until colonies formation. Colony shape of LSCs was observed under the light microscope, and colony-formation efficiency was analyzed after Giemsa staining by Image J software.
The morphology and density of corneal endothelial cells were normal in the corneal preservation solution with and without Y-27632 for 4 days. In the seventh day after preservation, the cells remained the regular hexagon in shape in the preservation solution with Y-27632, however, the cellular membrane was slightly shrinking with the positive staining for alizarin red in the preservation solution without Y-27632. The density of corneal endothelial cells in the corneal preservation solution without Y-27632 was (2 262±75) cells /mm2, while in the preservation solution with Y-27632 was (2 425±95) cells/mm2 (P<0. 001). The cloning spheres of LSCs were similar in preservation solution both with and without Y-27632 in the freshly isolated cornea or preserved corneas and exhibited more cells inside. But in 7 days and 14 days after preservation, the cloning spheres were much smaller in the preservation solution without Y-27632 group than those in the preservation with Y-27632 group. No significant differences were found in the cloning-formation rate and survival rate of corneal epithelial cells in corneas freshly isolated or preserved for 4 days in both groups (all at P>0. 05). In 7 days and 14 days after preservation, the active rates of corneal epithelial cells were (73. 00±2. 12)% and (56. 00±0. 71)% in the preservation solution with Y-27632, which were significantly higher than (66. 00±4. 00)% and (49. 00±0. 71)% in the preservation solution without Y-27632, showing statistically significant differences between them (t=3. 098, P=0. 018; t=9. 798, P=0. 000). In addition, the cloning-formation rates of LSCs were (11. 05±0. 21)% and (3. 10±1. 97)% in the preservation solution with Y-27632 in 7 days and 14 days after preservation, revealing significantly elevation in comparison with (2. 05±1. 20)% and (0. 40±0. 14)% in the preservation solution without Y-27632 (t=18. 107, P=0. 000; t=3. 184, P=0. 017).
Y-27632 promotes the vitality and cloning-formation ability of LSCs in corneal preservation medium, suggesting its potential use during storage of cornea.