The Effect And Mechanism On Bone Formation Of Bone Marrow Stromal Cell When Activating Wnt Signaling In The Osteocyte Of Aged Mice

ObjectiveAd-Cre was used to activate Wnt signaling in the osteocyte of aged Catnb mice,and Notch signaling in BMSCs and osteocyte of Rosa^Notch mice with bilateral flox modified stop cassette in front of NICD at R26 to explored the function and potential mechanism of Notch signaling in the context of wild type BMSCs coculturing with Wnt Signaling activated osteocyte of aged Catnb mice on bone formation by detecting whether activating Wnt signaling in the osteocyte of aged nice can promote bone formation or not,and the effect and mechanism of Notch signaling on osteogenic differentiation of different stages of osteoblastic cells in this study.Methods1.The osteocyte of aged Catnb mice?>18 months?,and BMSCs as well as osteocyte of Rosa^Notch mice were transfected with Ad-Cre and Ad-GFP,respectively.2.Wnt or Notch signaling activated osteocyte were cocultured with BMSCs of wildtype C57BL/6,respectively.3.ALP was tested by alkaline phosphatase staining and biochemical quantification.Alizarin red S staining was used to test calcium deposition on21d after specific osteogenic induction.4.Western-Blot was used to detect the expression of?-catenin,NICD,and Jag1 in osteocyte.Wnt signaling target genes?Lef1,Axin2,Smad6?,and Notch signaling target genes?Hes1,Hes5,Hes7,Hey1,Hey2,HeyL?,and osteogenic differentiation makers?ALP,OSX,OCN,Runx2,Col?,Bsp?,as well as Notch ligands?Jag1,Jag2,Dll1,Dll3,Dll4?were detected by qPCR.Results1.The expression of Wnt signaling target genes?Lef1,Axin2,Smad6?and?-catenin protein?P<0.05?increased Significantly,which suggested the activation of Wnt signaling in aged Catnb mice osteocyte in vitro.2.The increasing expression of Notch signaling target genes?Hes1,Hes5,Hes7,Hey1,Hey2,HeyL?and NICD protein in BMSCs and osteocyte?P<0.05?,which suggested the activation of Notch signaling in vitro.3.Wnt signaling activated osteocyte significantly enhanced the expression of osteogenic differentiation markers?ALP,OSX,OCN,Col?,Runx2?and Notch signaling receptor Notch1,and calcium deposition level on 21d of cocultured BMSCs.At the same time,osteogenic differentiation and calcium deposition level on 21d of Notch signaling activated bone marrow stromal cells were significantly increased.4.Activated Notch signaling osteocyte notably inhibits the expression of osteogenic markers?ALP,OSX,OCN,Col?,Runx2,Bsp?and calcium deposition level on 21d of cocultured BMSCs.The mRNA transcription level of Jag1 was significantly increased in Notch signaling activated osteocyte,while the mRNA transcription level of Dll4 was decreased significantly.The expression of Jag1 protein in Notch signal activated osteocyte was notably reduced after lentivirus of Crispercas9-mJag1 worked on it,Which significantly increased ALP staining and mRNA transcription level of osteogenic differentiation makers?ALP,OSX,OCN,Runx2,Bsp?of cocultured BMSCs?P<0.05?.Conclusion1.Activating Wnt signaling in the osteocyte of aged mice promote osteogenic differentiation and bone formation by activating Notch signaling of cocultured BMSCs.2.Notch signaling activated osteocyte repressing osteogenic differentiation and bone formation of cocultured BMSCs.