| Objective: By establishing an in vitro oxygen-glucose deprivation/reoxygenation(OGD/R)model to simulate hypoxia-ischemia(H-I)neurological diseases.To explore the protective effect and mechanism of miR-199a-5p in bone marrow mesenchymal stem cells exosome(BMSCs-Exo)on neural stem cells(NSCs).Seek new methods for the treatment of neurological diseases.Methods:(1)Extraction of primary neural stem cells from the hippocampus of embryonic SD rats and spinal cord of neonatal 1-day-old rats,and identify NSCs by immunofluorescence.BMSCs were cultured until P4-P6 was replaced with Exo-free serum,and the Exo in the cell supernatant was extracted by differential centrifugation.Western Blot method was used to determine Exo-labeled proteins CD9 and CD63,the size and morphology of Exo were observed under transmission electron microscope,and the particle size distribution and concentration of the extracted vesicles were determined by Exo nanoparticle tracking analysis(NTA).Label exosomes with the fluorescent dye Di O and co-culture with e NSCs.(2)Establish OGD/R model: Establish cell growth curves of OGD4 h,R24h,R48 h,R72h,R96 h,R120h in OGD/R group and Normal group by CCK-8 method.The extracted P2 generation NSCs were divided into four groups:normal group(Normal),oxygen glucose deprivation/reperfusion group(OGD/R),exosome group(Exo),miR-199a-5p overexpression exosome group(miR-199a-5p-Exo).The OGD/R model was established by OGD4 h,R48h,the number of viable cells in each group was detected by CCK-8 method,the expression of NSCs marker Nestin in each group was detected by immunofluorescence.Edu proliferation test detects the number of proliferating cells in each group.q RT-PCR method was used to detect the expression of miR-199a-5p in each group of cells.The dual luciferase experiment verifies the dual luciferase activity of miR-199a-5p combined with the wild-type or mutant 3’UTR region of GSK-3β.The expression of GSK-3β and β-catenin was detected by Western Blot method.Results:(1)Successfully extracted NSCs from the hippocampus of embryonic SD rats and spinal cord of neonatal 1-day-old rats and they became obvious suspension spheres under the microscope.The immunofluorescence results showed that the NSCs marker nestin was expressed.The BMSCs-Exo electron microscope showed a saucer-like vesicle structure.The NTA results showed that the average particle size was 130 nm,and the Western Blot results showed the expression of Exo surface markers CD9 and CD63.It can be seen under the microscope that BMSCs-Exo is swallowed by NSCs.(2)The OGD/R model of NSCs was successfully established.The CCK-8 results showed that OGD 4h,reoxygenation 24 h,cell survival rate decreased by 40%-60%,reoxygenation24h-48 h cell proliferation most.Immunofluorescence and CCK-8 results showed that the Exo group had a higher cell survival rate than the OGD/R group,and the overexpression miR-199a-5p-Exo group had the highest cell survival rate.Edu experimental results confirmed that Exo promotes Edu expression after OGD/R,and the expression of Edu in the miR-199a-Exo group is more than that in the Exo group.PCR results showed that the expression of miR-199a-5p in Exo group was higher than that in OGD/R group,and the expression of miR-199a-5p in miR-199a-5p-Exo group was the highest.Dual luciferase results confirm that GSK-3β is the direct target of miR-199a-5p.Western Blot results showed that Exo inhibited the expression of GSK-3β and increased the expression ofβ-catenin.The expression of GSK-3β in the miR-199a-5p-Exo group was the lowest,and the expression of β-catenin was the highest.Conclusions:(1)There are more NSCs in the hippocampus of embryonic SD rats than in spinal cords of neonatal 1-day-old rats,and the cell viability is better.The cell vesicles extracted by differential centrifugation are Exo.(2)BMSCs-Exo has a protective effect on NSCs after OGD/R.BMSCs-Exo overexpressing miR-199a-5p has a stronger effect on promoting the proliferation of NSCs than Exo.(3)miR-199a-5p can inhibit the expression of GSK-3β in the Wnt pathway and increase the expression of β-catenin,thereby promoting the proliferation of NSCs after OGD/R. |
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