Regulation Of Bone Formation By Osteocyte Jag1

Objective:Lentivirus CRISPR/Cas9 was used to construct osteocyte knockout Jag1 stable strain MLO-Y4-?Jag1,to study whether osteocytic Wnt signal promotes osteogenic differentiation of bone marrow stromal cells through Wnt-Jag1 pathway;LV-Jag1 was used to construct osteocyte overexpression Jag1 stable strain MLO-Y4-Jag1,to study the effect of osteocyte Jag1 on osteogenic differentiation of bone marrow stromal cells and its potential application potential in bone tissue engineering.The purpose of this study is to provide a theoretical basis for the research and development of drugs targeting osteocyte Jag1 to promote osteogenesis and bone tissue engineering to repair bone defectsMethod:1.MLO-Y4 Wnt signaling was activated by activator and co-cultured with ST2 in the medium containing Notch signal inhibitor DAPT to explore whether Notch signal mediates MLO-Y4 Wnt signal to promote osteogenic differentiation of ST22.p LV[g RNA]-EGFP,p LV[Exp]-CBh>h Cas9(ns)was transfected into MLO-Y4 to obtain Jag1 knockout stable strain MLO-Y4-?Jag1,and activate its Wnt signaling,then co-cultured with ST2 to explore whether MLO-Y4 promote osteogenic differentiation of ST2 through Wnt-Jag1 pathway.3.ML0-Y4 were infected by Ad-BMP4,and then co-cultured with ST2 cells in the medium containing Notch signal inhibitor DAPT to explore whether Notch signal mediates osteogenic differentiation of ST2 promoted by MLO-Y4-BMP4.4.LV-Jag1 and LV-Jag2 were respectively transfected into MLO-Y4 to obtain stable transgenic strains of MLO-Y4-Jag1 and MLO-Y4-Jag2,which overexpressed Jag1/Jag2 stably.Then,co-culture with ST2 to explore the effect of single Notch ligand Jagged in MLO-Y4 on osteoblastic differentiation of ST25.The application potential of osteocyte Jag1 in bone tissue engineering was explored though the functional modules containing MLO-Y4-Jag1 and ST2,constructed by integrated printing of cell and materials6.The ability of osteogenic differentiation was determined by ALP activity(ALP staining and biochemical quantification)and the expression of markers of osteogenic differentiation(ALP,OSX,OCN,Runx2,Col I,BSP).Results:1.Wnt-activated MLO-Y4 significantly promoted the osteogenic differentiation of ST2 in co-culture system.When Notch signal was inhibited,and the osteogenic differentiation of ST2 was decreased.2.The stable transgenic strain MLO-Y4-?Jag1 was successfully constructed by CRISPR/Cas9,the ability of Wnt-activated MLO-Y4 to promote osteogenic differentiation was reduced when Jag1 was knocked out in MLO-Y4.3.Ad-BMP4 successfully infected MLO-Y4,significantly promoted the osteogenic differentiation of ST2 in co-culture system,and reduced the osteogenic differentiation after Notch signaling was inhibited.4.The stable strain of MLO-Y4-Jag1,MLO-Y4-Jag2 which overexpressed Jag1/Jag2 was successfully constructed and co-cultured with ST2 cells.MLO-Y4-Jag1 promoted osteogenic differentiation of ST2 and inhibited Notch signal,resulting in decreased osteogenic differentiation,while MLO-Y4-Jag2 had no promoting effect on the osteogenic differentiation of ST2.5.The 3D printing of the MLO-Y4-Jag1 functional module promotes the osteogenic differentiation and mineralization of ST2,indicating its potential application in tissue engineering to repair bone defects.Conclusion:1.Wnt-activated osteocytes may promote osteogenic differentiation of bone marrow stromal cells through up-regulated Jag1 and BMP4,which may be related to the activation of classic Notch signaling.2.Osteocyte overexpression Jag1 promoted osteogenic differentiation of bone marrow stromal cells and showed its potential application in bone tissue engineering,while the overexpression of Jag2 by osteocytes did not promote osteoblastic differentiation of bone marrow stromal cells.