| Objective(1)To develop a laser-induced animal model of corneal endothelial dysfunction,and provide a study basis for the treatment of it.(2)To investigate whether Y-27632,an inhibitor of Rho-associated protein kinase(ROCK),could promote the proliferation of umbilical cord blood endothelial progenitor cells(EPCs),and the differentiation towards corneal endothelial cell(CEC)-like cells,and eventually replace the CEC to treat corneal endothelial dysfunction.(3)To preliminarily explore the role of Pten,a tumor suppressor gene,in the G1 arrest and migration of CEC,and find a new therapeutic target to promote the in situ corneal endothelial regeneration.Methods(1)Neodymium-doped yttrium aluminum garnet(Nd: YAG)laser was used to injure the corneal endothelium of rabbits in a uniform and dispersed manner.Slit lamp,optical coherence tomography,Goldmann applanation tonometer,and Heidelberg retinal tomography were used to evaluate the corneal transparency,edema and intraocular pressure,etc.;scanning electron microscopy and histological examination were used to evaluate the corneal endothelial injury,and assess the efficacy,safety and stability of the laser-induced model comprehensively.(2)EPC was cultured in conditioned medium with the supplement of Y-27632.The proliferation of EPC was evaluated by Brd U,Ki67 immunocytochemistry,and CCK-8 assay;the differentiation of EPC was detected by quantitative polymerase chain reaction,western blotting and immunocytochemistry;intracameral injection was used to repair the laser-injured animal model.During the follow-up,corneal transparency,edema,intraocular pressure and corneal endothelial wound healing were observed.(3)Western blotting was used to detect the expression of Pten in corneal endothelium from different species.Pten inhibitor was applied,and then cornea organ culture,Brd U staining,flow cytometry and immunocytochemistry were used to detect CEC cell cycle;cell migration and invasion assays were used to measure cell migration potential.Finally,to evaluate its effect on corneal endothelial wound healing,Pten inhibitor was injected into the anterior chamber of a rat model of corneal endothelial dysfunction.Results(1)With 3 m J Nd: YAG laser energy,a 9-mm diameter zone of the rabbit corneal endothelium was injured in a uniform and dispersed fashion,which was sufficient to make a rabbit model of corneal endothelial dysfunction.(2)Y-27632,not only promoted EPC proliferation,but also improved its differentiation efficacy towards CEC-like cells.Intracameral injection of combined Y-27632 and EPC could accelerate the recovery of cornea transparency,the regression of cornea edema and the healing of corneal endothelium,comparing to Y-27632 or EPC injected alone.(3)A difference of the Pten expression was detected between human and rat corneal endothelium,which was related to their different proliferation ability.The Pten inhibitor could relieve the TGF-β2 induced CEC G1 arrest through PI3K/Akt signaling pathway,and promote CEC proliferation and migration.Intracameral injection of Pten inhibitor could promote the corneal endothelial wound healing in a rat model.Conclusion(1)Nd: YAG laser was effective to develop a rabbit model of corneal endothelial dysfunction,with minor invasiveness,high accuracy,few side effects and good reproducibility.(2)Y-27632 combined with conditioned medium promoted the proliferation and differentiation of EPC towards CEC-like cells,and the combination was beneficial to the wound healing of corneal endothelium.(3)Inhibition of Pten activity improved corneal endothelial regeneration,and could be a promising new therapeutic target for corneal endothelial dysfunction. |
