The Roles And Mechanisms Of PAX6/CXCL14 Axis In Promoting Corneal Wound Healing

Background:Cornea is an important component of ocular surface barrier and refractive system.Cornea exposed to external environment is vulnerable to various forms of external injuries and pathological injuries.The incidence of corneal injury is high.Poor repair can cause corneal opacity,keratinization of corneal epithelium,formation of new blood vessels,and serious corneal blindness.Corneal repair is a very complex process.Its complete mechanism and potential genetic control have not been fully elucidated so far.At present,the mechanism of limbal stem cells contributing to the repair of corneal injury is relatively in-depth study,but the role of corneal epithelial cells in the repair of corneal injury is scarce.In order to explore the role of corneal epithelial cells in the repair of corneal injury and its mechanism,we carried out relevant research.Methods:1.Establish rat models of central corneal epithelial injury,peripheral corneal limbal epithelial injury and whole corneal epithelial injury,and observe the healing of corneal injury,corneal state and ultimate outcome of the three injury models.2.To evaluate the specific expression of CXCL14 in cornea by molecular bioinformatics analysis,gene data detection and analysis,and to verify the expression and localization of CXCL14 in cornea by immunohistochemistry,Western Blot and Real-time PCR.3.Construct stable cell lines of human corneal epithelial cells overexpressing CXCL14 and down-expressing CXCL14.After verifying the stability effect,the effects of CXCL14 on corneal epithelial cells were evaluated by cell cloning and cell cycle experiments.The corneal injury models of rats overexpressing CXCL14 and downexpressing CXCL14 were established and the effects of CXCL14 on corneal injury repair were evaluated.4.Analysis of genome sequencing data and immunocoprecipitation and blocking experiments.To search for CXCL14 receptors in corneal epithelial cells,downstream signaling pathways or target genes activated or regulated by CXCL14,and to study the mechanism of CXCL14 acting in cornea.5.To construct a stable cell line of human corneal epithelial cells with downexpressed PAX6,and to verify the regulatory effect of PAX6 on CXCL14 in cells and animals by genome sequencing and CXCL14 blocking reply test.6.To evaluate the effect of CXCL14 recombinant protein on corneal injury repair and its clinical application prospects.Results:1.CXCL14 is highly expressed in corneal epithelium,especially in basal cells of whole corneal epithelium.2.CXCL14 promotes the proliferation and self-renewal of corneal epithelial cells,thus making rat corneal epithelial cells play an important role in repairing corneal injury in the absence of limbal stem cells.CXCL14 promotes the repair of corneal injury by corneal epithelial cells and can maintain the stability of corneal tissue structure.3.CXCL14 can activate the downstream NF-kappa B signaling pathway maybe by binding with SDC1 receptor,thus promoting the proliferation of corneal epithelial cells,thereby promoting the repair of corneal injury and maintaining the stability of corneal tissue and structure.4.PAX6 directly regulates the expression of CXCL14 in corneal epithelial cells.PAX6 promotes the proliferation of corneal epithelial cells and the repair of corneal injury by regulating CXCL14.5.CXCL14 recombinant protein has the function of promoting the repair of corneal epithelial injury,and has no obvious toxic and side effects on corneal tissue and cells.Conclusions:1.Corneal epithelial cells play an important role in the repair of corneal injury.This repair function is determined by the biological characteristics of corneal epithelial cells.While paying attention to the repair function of limbal stem cells,the characteristics and functions of corneal epithelial cells should also be paid attention to.2.CXCL14 is highly expressed in corneal epithelial cells,and there is no significant difference in the expression of CXCL14 in corneal epithelial cells and limbal stem cells.CXCL14 is an important gene for corneal epithelial cells to repair corneal injury and maintain normal corneal homeostasis.3.PAX6 directly regulates CXCL14,and then CXCL14 may binds to SDC1 receptor to activate the downstream NF-kappa B signaling pathway,thereby promoting the proliferation of corneal epithelial cells,thereby promoting the repair of corneal injury and maintaining the stability of corneal tissue structure.4.Recombinant CXCL14 protein has remarkable repair function for corneal injury,and has no obvious toxic and side effects on corneal tissue and cell morphology,so it has good clinical application prospects.