In Vitro Experiments To Explore The Role And Mechanism Of Gli2 In The Interaction Between Exogenous Neural Stem Cells And Spinal Cord Endogenous Neural Stem Cells

Background:The recovery of Neural function after spinal cord injury has always been a difficult clinical problem.With the deepening of the understanding of neural regeneration-related signaling pathways,regulating the proliferation and differentiation of spinal cord endogenous neural stem cells has become a hot spot in recent years.Studies have shown that the Wnt/β-catenin signaling pathway activates and promotes the proliferation of spinal cord endogenous neural stem cells after spinal cord injury.In addition,there are also studies using Shh signaling pathway agonists in spinal cord injury animal models to reduce the area of inflammation,retain more white matter,and promote the proliferation and differentiation of neural precursor cells.Among them,Gli2 protein is the main effector of Shh signaling pathway.In recent years,some researchers have found that Gli2 protein and β-catenin,a key downstream factor of the Wnt pathway,mutually regulate and promote the activity of related pathways.Therefore,it is of great clinical significance to explore the mechanism of Wnt pathway of Gli2 spinal cord endogenous stem cells.Objective:To study the role of Gli2 in the influence of exogenous neural stem cells on the proliferation,differentiation and survival of spinal cord endogenous neural stem cells,and explore its possible mechanism of action.It provides an experimental basis for promoting the orderly proliferation and differentiation of endogenous neural stem cells,thereby achieving the regeneration and functional recovery of injured spinal cord anatomy.Methods:1.Extract neural stem cells of neonatal rat cerebral cortex and endogenous neural stem cells of adult rat spinal cord,and perform immunofluorescence identification of specific markers on the surface of neural stem cells.2.In the Transwell co-culture system,the CKK8 method was used to compare the effects of different concentrations of neural stem cells on the proliferation of spinal cord endogenous neural stem cells.Western blot and RT-PCR were used to verify the optimal co-culture concentration.3.Transfect the overexpressed and silenced Gli2 plasmid with the Nucleofector nuclear transfection instrument to transfect the exogenous cortical neural stem cells.4.Exogenous cortical neural stem cells overexpressing and silencing Gli2 were co-cultured with spinal cord endogenous neural stem cells at the optimal co-culture concentration,and the proliferation rate of endogenous stem cells in each group was compared by CCK8 method.Western blot and RT-PCR detected the key proteins of Wnt pathway,AXIN2 and DKK1,and simultaneously detected astrocyte GFAP and neuroprotective protein FOXO4.Results:1.The biological characteristics of spinal cord endogenous neural stem cells and cortical neural stem cells are roughly the same.They are all grown in suspension,resemble a circle,and are all spherical.Immunofluorescence staining nestin(+).2.The best co-culture concentration of exogenous cortical neural stem cells and spinal cord endogenous neural stem cells in Transwell co-culture system is 2:1.3.Exogenous cortical neural stem cells that overexpress Gli2 can enhance the cell proliferation ability of spinal cord endogenous neural stem cells,and the expression of activating factor AXIN2 of Wnt pathway is up-regulated,the expression of inhibitory factor DKK1 is down-regulated,astrocyte-specific marker GFAP The expression is up-regulated,and the expression of neuronal protective protein FOXO4 is up-regulated.Conclusion:This experiment proved that Gli2 may activate AXIN2 and inhibit DKK1 from activating the Wnt pathway,thereby enhancing the proliferation and cell viability of spinal cord endogenous neural stem cells,and promoting their division into astrocytes.