| Objective:In this study,diabetic rat model and high glucose cultured limbal stem cell were used to discover the effect of migration and proliferation of corneal limbal stem cells induced by high glucose environment.And investigate the role of substance P in diabetic corneal wound healing.Methods:1.Animal experiments:Establish 30 diabetic rats model,and divided the animals into two groups randomly:15 rats for diabetic group,15 rats for substance P group.Another 15 normal SPF rats were regarded as control group.All rats corneal epithelial central was scraped to observe the epithelial repair speed after wounding.The epithelial cell structre and messechymal molecule Vimentin,?-catenin and P63 expressions were detect by HE stain and immunohistochemial analysis.2.Cellular and molecular experiments:CLSCs were cultured wih high glucose(25mM)for 24h,then treated with substance P for 48h and cell migration and proliferation were measured by CCK-8,the protein expression level and intracellular location of vimentin,N-cadherin,?-catenin,MMP9,FAK and IGF-1 were measured by western-blot and immunofluorescene.The interactions between IGFBP and Iwere detected with immune co-precipitation to explore the protein factors that may interact with substance P in the signal transduction pathways.Results:1.The comeal epithelial healing retard in diabetic rats after the mechanical curettage of comeal epithelium,in the opposite,the corneal erosion of diabetic rats was enlarged after 24 hours.The epithelial layer became thinner and the structures were disorganized in diabetic rats through HE staining,and the columnar basal cells were reduced and the nucleolus shrinked and rounded.The immunohistochemical assay revealed the cell apoptosis and the arrest of migration were correlated with the lost expression of P-catenin,vimentin and P63 of cornea limbal stem cells.2.The proliferation and migration of human limbal epithelial cells were significantly decreased when exposed to high glucose,and the inhibition was accompanied by the decreased expression of P63.High glucose inhibit cell migration,spread and adhesiveness.The immunofluorescence staining and western-blot detection indicated the high glucose induced corneal stem cells transform to epidermal cells from mesenchymal cells.3.The corneal epithelial healing time of the substance P group was 72 hours which is significantly acceleraced compared with the diabetic group.The immunohistochemical analysis of cornea show the expression of Vimentin??-catenin and P63 were increased when using the eyedrop of substance P.After the treatment of substance P,the proliferation and migration were enhanced compared to diabetic group and the expression of the protein indicatd messenchymal cell and stemness were increased.4.The co-immunoprecipitation analysis demonstrated the vivo binding and functional relation between IGF-1 and IGFBP was decreased in SP group.And the concentration of single protein IGF-1 was increased.Conclusion:1.High glucose can significantly inhibit proliferation and migration of cornea limbal stem cells,it directly affects the interaction between basal cells and basement membrane in corneal epithelium.2.The depolymerization of IGFBP was accelerated after local application of substance P.Through the degeneration of IGFBP and the limbal stem cell damage can be partial repaired by substance P.3.The mechanism of substance P was related to the depolymerization of IGF-1 and IGFBP protein complexes.The releasing of free IGF-1 could increase the proliferation of limbal stem cells and accelerat the Epithelial-Mesenchymal Transition of high glucose cultured cells. |
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