| Objective: To observe the effect of BMSC Exos on osteogenesis in steroid-induced femoral head necrosis(SFHN),and to explore the related genes and their roles involved in this process.Materials and methods: BMSCs isolated from SFHN rats and healthy rats were used to isolate Exos from healthy rat-derived BMSCs using Exosome Precipitation Kit,and Exos was confirmed by transmission electron microscopy and Western-blot.SFHN rat BMSCs were randomly divided into two groups: model group and incubation group.Healthy rat BMSCs were used as control.Three groups of cells were co-cultured with adipogenic medium and osteogenic induction medium respectively.Oil red O staining analysis Adipogenic differentiation of three groups of cells;alizarin red staining method to evaluate the osteogenic differentiation of the three groups of cells;The Osteogenesis RT2 Profiler PCR Array was used to compare the differentially expressed genes(DEGs)in the model group and the incubation group,and the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis was performed using the DAVID 6.8 online software.Gene Ontology(GO)functional enrichment analysis,using STRING database to predict and analyze the interaction between DEGs-encoded proteins,using Cytoscape software to construct PPI network,screening target DEGs;The lentiviral-mediated si RNA vector of the target DEGs was transfected into three groups of cells,and the cell transfection rate was detected by Western-blot and real-time quantitative PCR.The lentiviral-mediated si RNA vector of the target DEGs was transfected into three groups of cells.The cell transfection rate was detected by Western-blot and real-time quantitative PCR.The cells were co-cultured with adipogenic medium and osteogenic induction medium.The red o staining method was used to analyze the adipogenic differentiation of cells,and the alizarin red staining method was used to evaluate the osteogenic differentiation of cells.Results: The results of transmission electron microscopy(TEM)showed that the particles obtained from BMSCs were 100-150 nm in size,uniformly distributed,and had a complete membrane structure.Western-blot results showed that the particles significantly expressed CD63 protein.Compared with the control group,the number of lipid droplets and the area of alizarin red staining decreased significantly in the model group(P < 0.01).Compared with the model group,the number of lipid droplets and the area of alizarin red staining increased significantly in the incubation group(P < 0.01).Compared with the model group,there were 20 differentially expressed genes in the incubation group,including 11 up-regulated genes and 9 down-regulated genes.Bioinformatics analysis showed that DEGs were mainly involved in immune response,osteoblast differentiation and transforming growth factor-beta/bone morphogenetic protein signaling pathway.PPI network indicated that Sox9 was up-regulated and interacted with protein.Use closely.Western-blot and real-time quantitative PCR results showed that compared with non-transfected BMSCs,the expression of Sox9 protein and gene in cells transfected with lentivirus-mediated Sox9-si RNA was significantly lower(P < 0.05);compared with control cells,the expression of Sox9 protein in model cells was significantly lower(P < 0.05),and compared with model group,incubation was significant(P < 0.05).Compared with non-transfected BMSCs,the number of lipid droplets and the area of alizarin red staining in transfected BMSCs were significantly increased and the area of alizarin red staining was significantly reduced(P < 0.05).Conclusions: BMSCs Exos can promote the osteogenesis of SFHN.In the process of BMSCs Exos promoting SFHN osteogenesis,20 kinds of DEGs were detected,11 of which were up-regulated and 9 were down-regulated.These changes may be related to altered immune responses and BMP/TGF-β signaling pathways.Sox9 is a key gene fragment regulating SFHN osteogenesis,which can promote its osteogenesis and affect the therapeutic effect of BMSC Exos on SFHN. |
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