Study On Related Mechanisms Of Osteoglycin On Osteoblastogenesis From Mesenchymal Stem Cells

Osteoporosis is characterized by low bone mass,degeneration of bone tissue and destruction of bone microstructure,which increases the risk of fracture.The underlying cause of osteoporosis is the imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption in the bone marrow microenvironment.Bone marrow mesenchymal stem cells play an important role in bone development,remodeling and regeneration by differentiating into osteoblast precursors.OGN has been shown to promote osteoblast precursors differentiating into osteoblasts,as well as osteoblast maturation and bone formation.Therefore,to study the role of OGN in the regulating osteoblastogenesis of bone marrow mesenchymal stem cell is of great clinical significance.Osteoporosis is a metabolic disease.The increase of fat in bone marrow caused by lipid metabolism disorder may cause bone marrow microcirculation disorders and bone loss.Fatty acids are an important component of lipids.Fatty acids and their oxidative metabolites,eicosanoic acids,are involved in many physiological processes and disease processes.Metabolomics can directly reflect the biochemical process and state changes of substances in vivo.Since the osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells are interrelated and interact on each other,it is of great significance to study eicosanoic acid during osteogenic differentiation of bone marrow mesenchymal stem cells induced by OGN from the perspective of metabonomics.Overall,this study firstly studied the expression of OGN during osteogenic differentiation of the mouse bone marrow mesenchymal stem cells.Secondly,we studied the effect of increased OGN expression on osteogenic differentiation of the cells.Finally,we analyzed the expression of eicosanoic acids during osteogenic differentiation of the cells after transfection of OGN-infected lentivirus based on Ultra Performance Liquid Chromatography(UPLC)and Triple Quadruple Mass Spectrometry system.In the first section,the proliferative capacity of mouse bone marrow mesenchymal stem cells was tested by MTT and clone colony assay.The cells were cultured in osteogenic and adipogenic differentiation medium,and the differentiation potential was verified by ALP staining and oil red O staining.The expression of OGN and PPARγ2 mRNA in osteogenic and adipogenic differentiation was detected.It was found that the expression of OGN mRNA was only significantly increased during osteogenic differentiation,while the expression of PPARγ2 mRNA was only significantly increased during adipogenic differentiation.Rosiglitazone was then used to treat the cells in osteogenic and adipogenic differentiation medium.It was proved to promote adipogenic differentiation by oil red O staining and detection of aP2 mRNA expression,and inhibit osteogenic differentiation by ALP staining.The expression of OGN and PPARγ2 mRNA decreased and increased respectively after rosiglitazone treatment.These results suggest that OGN plays an important role in osteogenic differentiation of mouse bone marrow mesenchymal stem cells.In the second section,we studied the ability of OGN to promote osteogenic differentiation of mouse bone marrow mesenchymal stem cells.The OGN gene was integrated into the lentivirus plasmid vector to transfect the cells.By alizarin red and ALP staining,it was found that the calcium deposition and calcium nodules were significantly higher in the transfected group than those in the control group.The expression of osteogenetic markers OGN,ALP,RUNX2,OCN and Wnt5 b were significantly increased after transfection of OGN-infected lentivirus,while osteoclast marker Rankl and adipogenic markers PPARγ2 and aP2 decreased significantly.These results proved that increased expression of OGN could promote the osteoblastogenesis of mouse bone marrow mesenchymal stem cells.In the third section,we tested the expression of 25 eicosanoic acids and 10 deuterated eicosanoic acids.The standard curve of eicosanoic acid was established.The expression of 25 eicosanoic acids in mouse bone marrow mesenchymal stem cells at different time points in osteogenic differentiation was studied.We found the expression of DHA and AA reduced significantly as the time went on.DHA plays an important role in the biological function of osteoblasts.AA was proven to promote the adipogenic differentiation of mesenchymal stem cells.The effect of OGN on promoting osteoblastogenesis of mouse bone marrow mesenchymal stem cells was further verified.