Dexamethasone Induce The DeSUMOylation Of Neural Stem Cells And Decreases The Stemness Of Neural Stem Cells

Purpose:1.Observe the forms of SUMO1 in NSCs.2.Observe the effects of DEX on the proliferation and differentiation of NSCs.3.Observe the effects of DEX on the SUMOylation of NSCs.4.To investigate whether DEX can decreases the stemness of NSCs and induce the de SUMOylation of NSCs.Research content and methods:1.The forms of SUMO1 in NSCs.The striatum was dissected from embryonic day 14.5 C57BL/6 mice by cesarean section under sterile conditions and cultured for primary culture.The primary cells were identified by immunofluorescence staining.Fetal bovine serum(FBS)was used as a positive control.First,we observed cell aggregates and morphological changes under a microscope.Nest,we double immunostained the cells for Nestin??3-tubulin?GFAP?SUMO1 and Oct4.Western Blot analysis was performed to detect the level of Oct4 and SUMO1.2.The effects of DEX on the proliferation and differentiation of NSCs.NSCs were treated with DEX for 48 hours and observed cell aggregates and morphological changes under a microscope.CCK8?Edu and immunofluorescence staining were performed to observe the effects of DEX on the proliferation and differentiation of NSCs.3.The effects of DEX on the SUMOylation of NSCs.NSCs were treated with DEX for 48 and 96 hours.Then Western Blot analysis was performed to detect the level of SUMO1?Oct4?Nestin?MAP2 and some SENPs in NSCs.Results:1.In undifferentiated nerve cells,SUMO1 exists mainly in a combined form rather than a monomeric form.Immunofluorescence staining showed that the immunoreactivity of Nestin?Sox2and Oct4 was strongly positive in neurospheres in NSC medium,which confirmed the primary cells we dissected from striatum were NSCs.The NSCs were adherent and mostly differentiated prematurely into nerve cells after exposure to 1% FBS for 48 hours compared with the untreated control group.Immunoreactivity of nestin was strongly positive,whereas ?3-tubulin was barely detected in neurospheres.In contrast,GFAP and ?3-tubulin double-positive cells showing typical neuronal morphology were obviously increased.In addition,the immunoreactivity of SUMO1 and Oct4 in the nucleus was decreased,whereas the immunoreactivity of SUMO1 in the cytoplasm was increased.Western Blot showed that SUMO1 ylated proteins were decreased by 1% FBS,and the monomer form of SUMO1 was increased.2.DEX decreases the proliferation of neural stem cells and induces differentiation.NSCs were treated with DEX.The observation under a microscope revealed that monoclonal NSC spheres could not maintain floating multicellular structures.CCK8 and Edu assay indicated that DEX decreased the proliferation of NSCs.Next,Immunofluorescence staining showed that after exposured to DEX for 48 and 96 hours,morphological changes were obvious,even single neurons was seen after 96 hours.Moreover,after treatment with DEX for 96 hours,the number of?3-tubulin-positive cells was higher than at 48 hours.These results suggest that DEX induces NSC differentiation that may be time dependent.3.DEX decreases SUMO1-conjugated proteins and induces de SUMOylation of NSCs.Western blot analysis indicated that SUMO1 modification proteins were indeed decreased after NSCs were treated with DEX for 48 and 96 hours,SENPs,as desumoylation enzymes,were also detected.SENP5 showed an increase in expression(P<0.05).Interestingly,the expression of SUMO1-conjugated Oct4 was decreased,Moreover,MAP2 showed a time-dependent increase in expression.Conclusion:Dexamethasone Induce the de SUMOylation of Neural Stem Cells and Decreases the Stemness of Neural Stem Cells.