| Summary: | Objective To investigate the effect and underlying mechanism of activating Notch signaling in osteocytes on osteogenic differentiation of bone marrow stromal cells (BMSCs). Methods The osteocytes derived from RosaNotch mice were infected with recombinant adenovirus expressing Cre or GFP respectively, and then the obtained cells were co-cultured with BMSCs isolated from wild-type C57BL/6 mice, and named as Ad-Cre group and Ad-GFP group, with a BMSCs group without any treatment as blank control. Alkaline phosphatase (ALP) staining and detection of ALP relative activity were used to measure the expression of ALP. The mRNA expression levels of the target genes in osteocyte of RosaNotch and ligands of Notch signaling, osteogenic markers, and angiogenic makers in the co-cultured product were detected by qPCR. Alizarin red staining was applied to test the matrix mineralization on day 21. Results Infection of recombinant adenovirus Ad-Cre could successfully activate Notch signaling. ALP staining and detection of ALP activity showed that the expression level of ALP was significantly lower in the Ad-Cre group than the Ad-GFP group and Blank group (P < 0.05), and there was no statistical difference in the level between the latter 2 groups. The results of qPCR indicated that the mRNA levels of osteogenic makers ALP, Osterix, Runx2 and Notch ligand Dll4 were deceased significantly, while those of Jag1, VEGF, hypoxia-inducible factor 1α (HIF1α), platelet endothelial cell adhesion molecule-1 (CD31/PECAM-1), and endomucin (EMCN) were statistically increased in the Ad-Cre group when compared with the Ad-GFP group and Blank group (P < 0.05). Alizarin red staining displayed that the Ad-Cre group had more calcium deposition in 21 d after co-culture than the Ad-GFP group and blank group (P < 0.05). Conclusion Activating Notch signaling in osteocytes inhibits osteogenic differentiation of BMSCs in vitro.
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