[Activation of Notch signaling in bone marrow stromal cells promotes osteogenesis in mice in vitro]

Objective To investigate the osteogenic differentiation of bone marrow stromal cells (BMSC) with Notch signaling activation in vitro. Methods The BMSC derived from Notch1-NICD^flox/flox mice were infected with recombinant adenovirus expressing Cre or GFP respectively, and designated as Ad-Cre group and Ad-GFP group. The expression of Notch1 was evaluated by Western blot analysis. Alkaline phosphatase (ALP) activity was determined by ALP staining and biochemical quantification, and the calcium deposition was analyzed with Alizarin red S staining. Real-time fluorescence quantitative PCR (qPCR) was used to quantify the expression of Notch target genes(Hes1, Hey1, Hey2, HeyL), osteogenic differentiation-related genes(ALP, RUNX2, osterix, osteocalcin), and angiogenesis factors(VEGF, HIF-1α). Results Notch signaling in BMSC of Notch1-NICDflox/flox mice was activated successfully by Ad-Cre, as was evidenced by the significantly elevated expression of Notch1 and Notch target genes. Compared with the Ad-GFP group, ALP activity and the late calcium deposition were significantly increased upon treatment with Ad-Cre. qPCR results demonstrated that the expression of ALP, RUNX2, osterix, osteocalcin, VEGF, HIF-1α in the Ad-Cre group were significantly upregulated. Conclusion Activation of Notch signaling in BMSC in vitro significantly promotes osteogenic differentiation.