Abstract
Transplantation of ex vivo cultured limbal epithelial cells is proven effective in restoring limbal stem cell deficiency. The present study aimed to investigate the promoting effect of Y-27632 on limbal epithelial cell proliferation. Limbal explants isolated from human donor eyes were expanded three weeks on culture dishes and outgrowth of epithelial cells was subsequently subcultured for in vitro experiments. In the presence of Y-27632, the ex vivo limbal outgrowth was accelerated, particularly the cells with epithelial cell-like morphology. Y-27632 dose-dependently promoted the proliferation of in vitro cultured human limbal epithelial cells as examined by phase contrast microscopy and luminescent cell-viability assay 30 hours after the treatment. The colony forming efficacy determined 7 days after the treatment was enhanced by Y-27632 also in a dose-dependent manner. The number of p63- or Ki67-positive cells was dose-dependently increased in Y-27632-treated cultures as detected by immunofluorescent staining and western blotanalysis. Cell cycle analysis by flow cytometric method revealed an increase in S-phase proliferating cells. The epithelial woundclosure rate was shown to be faster in experimental group received topical treatment withY-27632 than the sham control using a rat corneal wounding model. These resultsdemonstrate that Y-27632 can promote both the ex vivo and in vitro proliferation oflimbal epithelial cell proliferation. The in vivo enhanced epithelial wound healingfurther implies that the Y-27632 may act as a new strategy for treating limbal stem cell deficiency.
Highlights
The ocular surface is covered by corneal, limbal, and conjunctival epithelial cells that, together with a stable pre-ocular tear film, maintain its integrity
The corneal epithelium exists in a state of dynamic equilibrium, with the superficial epithelial cells being constantly shed into the tear pool
Proliferation of limbal epithelial stem cells is the first step to resurface the corneal epithelium in response to any ocular surface insults
Summary
The ocular surface is covered by corneal, limbal, and conjunctival epithelial cells that, together with a stable pre-ocular tear film, maintain its integrity. The corneal epithelium exists in a state of dynamic equilibrium, with the superficial epithelial cells being constantly shed into the tear pool. The cells shed from the corneal surface are replaced through proliferation of a distinct subpopulation of cells located at limbal basal layer, known as limbal stem cells (LSCs) [1]. Severe damage to the limbal epithelial cells from various etiologies in the limbal region may lead to loss of the limbal epithelial cells [2], so called limbal stem cell deficiency (LSCD). LSCD, manifested by chronic inflammation, neovascularization, and goblet cell invasion into the cornea, may be complicated by persistent corneal epithelial defects, ulceration, and even perforation of the cornea [3, 4]. The cornea may be healed by fibrosis, the vision will be greatly impaired
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