| BackgroundOsteoporosis(OP)is a systemic bone metabolic disease caused by a variety of factors.Most patients with osteoporosis are characterized by bone pain and fracture.The main cause of osteoporosis is the breakdown of the balance between osteoblasts and osteoclasts.Its main characteristic is the reduction of bone tissue volume per unit volume.Insufficient bone formation and weakened osteogenic differentiation are important factors in the pathogenesis of osteoporosis.Neural cell adhesion molecule(NCAM)is widely expressed in neural cells,and its function in the nervous system is more studied.In recent years,it has been found that NCAM is also expressed in osteoblasts and mesenchymal stem cells,but its biological significance is not fully understood.NCAM may be involved in the regulation of stem cell differentiation.Studies have shown that NCAM is involved in the maintenance of differentiation of bone marrow mesenchymal stem cells(BMSCs)into adipocytes,but the role and mechanism of NCAM in osteoblast differentiation is not yet clear.The goal of this project is to clarify the role and mechanism of NCAM in the regulation of osteoblast differentiation.Objective1.The effects of NCAM on osteoblast differentiation were determined by studying osteogenic precursor cells(MC3T3-E1)and BMSCs;2.The effects of NCAM on osteoblast differentiation is influenced by which signal pathway.Methods1.The changes of calcium deposition and NCAM were detected after adding specific osteoblast inducer to 0 d,3 d,7 d,and 14 d in the control MC3T3-E1 cells;2.NCAM silencing plasmid was transferred to normal MC3T3-E1 cells.Western blot and qPCR were used to detect the changes of RunX2,Osterix and calcium deposition.3.The culture and differentiation of cells and the establishment of NCAM knockout BMSCs(KO cells)in vitro;4.The osteoblast markers RunX2,Osterix,and calcium deposition were detected by Western blot and qPCR techniques after the differentiation of osteoblasts in WT and KO cells for 0 d,3 d,7 d,and 14 d;5.The Wnt protein kinase specific inhibitor XAV939 and activator LiCl were added to the control and NCAM interfered MC3T3-E1 cells.Western blot,qPCR and alizarin red staining were used to detect the changes of RunX2,Osterix and calcium deposition.Results1.Previous studies have shown that in the process of osteoblast differentiation induced by MC3T3-E1,the expression of NCAM is significantly increased with the increase of osteoblast differentiation days.To clarify the effect of NCAM on osteoblast differentiation,we silenced the NCAM gene in MC3T3-E1.We found that RunX2 and Osterix markers were significantly down-regulated after NCAM was silenced,and calcium deposition was also significantly reduced,indicating that NCAM silencing can significantly inhibit osteoblast differentiation;2.To verify the effect of NCAM on osteogenic differentiation,we used NCAM knockout BMSCs(KO cells)in vitro.In normal BMSCs(WT cells),it was also found that the expression of NCAM was significantly upregulated with the increase of osteoblast differentiation days.When NCAM was deleted,the mRNA and protein expression levels of RunX2 and Osterix markers of osteoblasts were significantly down-regulated,and calcium deposition was significantly reduced.This shows that the deletion of NCAM can inhibit the differentiation of BMSCs into osteoblasts;3.In order to explore the molecular mechanisms underlying the inhibition of NCAM deletion in osteoblast differentiation,we investigated the signaling pathways involved.The results showed that the expression of β-cateninnin was significantly reduced after the NCAM deletion,and after inhibiting or activating the Wnt signaling pathway,the osteoblast marker RunX2 and Osterix were significantly down-regulated or up-regulated.Inhibition of osteoblast differentiation by NCAM deletion is achieved through the Wnt signaling pathway.Conclusion1.The deletion of NCAM inhibited the differentiation of bone cells;2.NCAM Wnt pathway is involved in the regulation of osteoblast differentiation. |
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