Effects Of BMP-7on Osteogenic Differentiation Of Prenatal Mouse NP Cell And Notch Signaling Pathway

ObjectiveTo investigate the effects of recombinant adenovirus-mediated bonemorphogenetic protein-7(Ad-BMP-7) over expression on osteogenicdifferentiation of prenatal mouse intervertebral disc nucleus pulposus(NP)cell, and explore whether BMP-7regulates classic Notch signal pathwayin this process.MethodsRecombinant Ad-BMP-7, Ad-GFP, Ad-Jag-1and Ad-RFP wereamplified by HEK293cell and used to infect NP cells. RT-PCR wasperformed to detect the mRNA expression level of BMP-7, Real-time PCRwas carried out to detect the mRNA expression level of osteogenesis indexOsteopontin(OPN), Osteoprotegerin(OPG) as well as the classic Notchsignal pathway members. Western blot was used to detect the proteinexpressions level of osteogenesis transcription factor Runx2and OPN.Alkaline phosphatase(ALP) activity was measured by using ALP assayand staining.ResultsThe high titers Ad-BMP-7, Ad-GFP, Ad-Jag-1and Ad-RFP weregenerated by adenovirus amplification in HEK293. The basic mRNAexpression level of BMP-7was very almost undetectable in NP cell. Compared with control group, Ad-BMP-7mediated over-expression ofBMP-7significantly increased the mRNA expressions level of the OPN,OPG and the protein expressions level of osteogenesis transcription factorRunx2, OPN (OPN：P=0.000，OPG：P=0.012). The activity of ALP, aspecific indicator for steogenesis in Ad-BMP-7group was statisticallyhigher than the control group (P=0.000), with enhanced positivepurple-blue stained cells. After3days of Ad-BMP-7infection, the mRNAexpression of classic Notch signal pathway members Notch-1, Notch-2,Jag-1, Hey-1significantly increased (Notch-1：P=0.002，Notch-2：P=0.000，Jag-1：P=0.000，Hey-1：P=0.000), while reversed to the baselineat day7. There was no difference of others Notch signal pathway membersamong treated groups. Activated the notch signaling pathway by Ad-Jag-1,the protein of Runx2and OPN expressions level and ALP activity inAd-BMP-7+Ad-Jag-1group were significantly higher than the controlgroup (P=0.000).ConclusionAdenovirus-mediated BMP-7over-expression can induce osteogenicdifferentiation of NP cell, which partly due to transient up-regulation ofsome classic Notch signal pathway members; activating the notchsignaling pathway can further promote BMP-7-induced osteogenicdifferentiation of NP cell.