| Objective: To explore the effects of bone marrow mesenchymal stem cells on apoptosis and autophagy in rat primary nucleus pulposus cells.Methods: Rat nucleus pulposus cells and bone marrow mesenchymal stem cells were isolated and cultured in vitro for co-culture experiments.The pathological state of low blood supply to the intervertebral disc was simulated by serum deprivation experiments.The apoptosis rate of rat nucleus pulposus cells was detected by apoptosis staining kit and flow cytometry.The apoptosis protein active-caspase3 and active-casepase 9 were detected by Western blot.Expression of proapoptotic protein Bax and apoptosis inhibitory protein Bcl2 protein.Subsequently,the expression of autophagy marker proteins LC3 and Beclin 1 was detected by Western blot.Results: The autologous and apoptotic levels of rat primary nucleus pulposus cells cultured in vitro were lower in serum.Co-culture of rat nucleus pulposus cells with bone marrow mesenchymal stem cells can significantly increase the level of autophagy in cells.After deprived of serum for 24 hours,the cell viability of rat primary nucleus pulposus cells was significantly reduced,and the level of apoptotic autophagy was significantly up-regulated.At the same modeling time,the viability of rat nucleus pulposus cells co-cultured with bone marrow mesenchymal stem cells increased,while the level of autophagy increased significantly and the level of apoptosis decreased significantly.However,when autophagy induced by bone marrow mesenchymal stem cells was inhibited by Wortmannin,the apoptotic rate was significantly increased.Conclusion: Bone marrow mesenchymal stem cells can significantly increase the level of autophagy in rat nucleus pulposus cells.In the serum deprivation experiment,bone marrow mesenchymal stem cells can significantly increase the cell viability,autophagy level and decrease the apoptosis level of rat nucleus pulposus cells.After Wortmannin inhibited autophagy induced by bone marrow mesenchymal stem cells,the cell viability of rat nucleus pulposus cells was significantly reduced,and the level of apoptosis was significantly increased.Objective: To explore the mechanism of bone marrow mesenchymal stem cells inducing autophagy in rat primary nucleus pulposus cells.Methods: Differential expression was screened by gene expression profiling to screen differentially expressed mi RNAs with fold change greater than 2.5 and FDR values ??less than 0.01 as a threshold.q RT-PCR method was used to detect the effect of different treatment methods on the expression level of mi R155.Flow cytometry and transmission electron microscopy were used to detect the effect of anti-mi R-155 on autophagy.The direct interaction between mi R-155 and BACH1 m RNA was detected using Targetscan software and luciferase reporter assay,and further validation was performed using Western blot.Result: A total of 15 differentially expressed mi RNAs were extracted by gene expression profiling.Among them,the expression difference of mi R-155 was the largest compared with NPC+OGD group.Therefore,mi R-155 was selected as a research object for further research.Compared with the OGD+NPC group,the expression level of mi R-155 in the OGD+NPC+BMSC group was significantly increased.The BMSC cells successfully transfected with anti-mi R155 were co-cultured with NPC cells.The results showed that the expression of mi R-155 was significantly decreased in the co-cultured cells transfected with anti-mi R155 compared with OGD+NPC+BMSC cells.In addition,we also blocked the secretion of exosomes of BMSC cells using GW4869(10 μM),and the results showed that the expression of mi R-155 was significantly decreased in the co-cultured cells.Transmission electron microscopy and flow cytometry studies showed that anti-mi R155 significantly reduced BMSC-induced autophagy in NPC cells.It was predicted by bioinformatics software Targetscan that BACH1 may be a potential target for mi R-155,as shown in Figure 5A.In order to further confirm the interaction between mi R-155 and BACH1 gene,we used luciferase reporter assay to detect the response of wild-type BACH1 gene and mutant BACH1 gene to mi R-155 and mi R-con in NPC cells.The fluorescence response intensity of the rat spinal cord primary neuronal cell line transfected with mi R-155 withwild-type BACH1 gene was significantly decreased,while the fluorescence response intensity of the mutant BACH1 gene NPC cells did not change significantly.It was pointed out that mi R-155 negatively regulates the expression of BACH1 by binding to the 3’UTR region of BACH1 m RNA.Conclusion:(1)BMSC cells increase the expression level of mi R155 in NPC cells by the exosome pathway.(2)mi R155 plays a role in promoting autophagy in BMSC-induced autophagy in NPC cells.(3)mi R-155 negatively regulates the expression of BACH1 by binding to the 3’UTR region of BACH1 m RNA.mi R-155 may affect autophagy through BACH1. |
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