Effects Of CoCl₂-Induced Hypoxia And Hypoxia-Reperfusion On The Expression Of SUMO Family Members In Mouse Neural Stem Cells

Objectives To establish hypoxia and hypoxia-reoxygenation(I/R)cell models of primary neural stem cells by extracting and culturing primary neural stem cells(NSC)to detect hypoxia and deficiency.Effect of oxygen reoxygenation on proliferation and apoptosis of neural stem cells.Furthermore,the expression of SUMO family members in protein and mRNA levels during hypoxia and hypoxia after hypoxia was detected,and the nucleus shift of SUMO1 and SUMO2/3 in this process was also detected.To further understand the role and mechanism of SUMO modification in reducing brain ischemia-reperfusion injury,in order to find a new treatment for ischemic brain injury.Methods C57BL/6 mouse primary neural stem cells were extracted and cultured by primary cell extraction and culture techniques.Primary neural stem cells were cultured by immunofluorescence(IF)double staining technique and Western Blot(WB).Identification.The chemical hypoxia inducing agent cobalt chloride(CoCl2)was selected as the hypoxiainducing agent of neural stem cells,and the model of hypoxia and hypoxia-reoxygenation injury of neural stem cells was established.CCK-8 and EdU assays the proliferative capacity of neural stem cells during hypoxia and hypoxia-reoxygenation;Western blot analysis of cysteine protease 3(Caspas-3)in neural stem cells under hypoxia and hypoxiareoxygenation Expression in.Western blot analysis of SUMO family members(SUMO1,SUMO2/3,UBC9,SENP1,SENP2,SENP3,SENP5,SENP6,SENP7)expression changes in protein levels during neural stem cell hypoxia and hypoxia-reoxygenation,polymerase chain The expression of mRNA levels in SUMU family members during hypoxia and hypoxia-reoxygenation of neural stem cells was detected by Polymerase Chain Reaction(PCR).The cytoplasmic nuclear fractionation technique was used to detect SUMO1 and SUMO2/3 in neural stem cells under hypoxia.And the pulp core shift during the process of hypoxia and reoxygenation.All Western blot and PCR results were quantified using ImageJ,and GraphPad Prism 7 and SPSS 24.0 software were used to analyze the data.In all experiments,the results of 3 sets of parallel experimental data were expressed as mean ± SD.The comparison between the two groups was performed by independent sample t test.P ≤ 0.05 indicated that the difference was statistically significant.Results (1)Primary neural stem cells grow in suspension under the microscope and can be stably subcultured under low algebra.Dry maintenance related protein Nestin and RNA binding protein 1(Musashi-1)are highly expressed in neural stem cells,differentiation-related proteins glial fibrillary acidic protein(GFAP),β-tubulin(β3-Tublin),micro The tubeassociated protein(MAP2)was down-regulated in neural stem cells,and the expression of neural stem cell marker proteins Nestin,SOX2,and Oct-2 was positive by fluorescent double staining.(2)The proliferation of neural stem cells decreased after hypoxia and hypoxiareoxygenation in the process of chemical hypoxia and hypoxia-reoxygenation induced by cobalt chloride(P < 0.05),and the proliferation of neural stem cells decreased further after hypoxia-reoxygenation(P < 0.05).The expression of activated Caspase-3 continued to increase during this process(P < 0.05).(3)The expression of SUMO2/3 was decreased in neural stem cells induced by cobalt chloride(P<0.05),and the expression of SUMO2/3 was decreased after reoxygenation(P<0.05).There was no significant change in the expression of SUMO1 during this process.P>0.05).(4)The expression of SENP1 and SENP2 in the process of hypoxia and hypoxia-reoxygenation induced by cobalt chloride was significantly increased(P<0.05),and SENP3,SENP5,SENP6 and SENP7 did not change significantly during the process(P<0.05).P > 0.05).(5)The mRNA levels of SENP1 and SENP2 in neural stem cells during hypoxia and hypoxia-reoxygenation were significantly increased(P < 0.05).(6)SUMO2/3 enters the nucleus after hypoxia in neural stem cells,and nucleus after reoxygenation(P < 0.05).Conclusions (1)Primary neural stem cells are scattered in suspension sphere growth and can be stably passaged under low algebra;(2)Neural stem cells proliferate during hypoxia and hypoxia-reoxygenation,and apoptosis increases.(3)The combined expression of SUMO2/3 increased after hypoxia in neural stem cells,and decreased after hypoxiareoxygenation,and the nucleus shift occurred.After hypoxia,it entered the nucleus and renucleated and nucleated;(4)SENP1 SENP2 expression in protein and mRNA levels increased during hypoxia and hypoxia-reoxygenation of neural stem cells.Figure [7],Table [3],Reference [123]...