Function And Mechanism Research Of Dickkopf-1 In BMP9-induced Osteoblast Differentiation Of C3H10T1/2 Mesenchymal Stem Cells

Part one: Recombinant adenoviruses BMP9 and Dkk1amplification and effective transfection ofmesenchymal stem cellsObjectives: Recombinant adenoviruses were designated Ad GFP,Ad Dkk1, and Ad BMP9(also expressing GFP),and we transfect the recombinant adenoviruses into HEK293 cells to generate the high titre adenovirus. Make sure whether the recombinant adenovirus could infect the C3H10T1/2 mesenchymal stem cells effectively and the appropriate dose of virus infection, and verify the purpose gene expression in stem cells.Methods: Transfection into HEK293 cells to obtain high titre recombinant adenovirus Ad-BMP9 and Ad-DKK1. And detect the purpose gene RNA and protein expression level in stem cells infected by recombinant adenoviruses via PCR and Western blot.Results: We get high titre recombinant adenovirus by HEK293 cells,which could infected C3H10T1/2 mesenchymal stem cells effectively. Wecould also observe the expression of green fluorescent protein in inverted fluorescence microscope, and taking 50% stem cells infected by the recombinant adenovirus as appropriate experimental dose. And detect the purpose gene RNA and protein expression level in stem cells infected by recombinant adenoviruses via PCR and Western blot. And confirm the effectiveness of the Ad-BMP9 and Ad-DKK1 by PCR and Western blot.Conclusions: We can obtain high titre recombinant adenovirus Ad-BMP9 and Ad-DKK by HEK293 cells, which infected C3H10T1/2mesenchymal stem cells effectively.And the further research could be performed with the recombinant adenovirus.Part two: The influence of Dickkopf-1 onBMP9-induced Osteoblast Differentiation ofMesenchymal stem cellsObjectives: To investigate the potential role of Wnt signaling antagonist Dickkopfl(DKK1) on BMP9-induced ostoegenic differentiation of MSCs.Methods: C3H10T1/2 cells were infected by Ad-BMP9 andAd-DKK1.The ALP was detected by ALP activity assay and staining,the expression of OC and OPN were detected by RT-PCR and Western blotting and calcium deposition was determined by Alizarin Red S staining.Meanwhile, Ectopic bone formation assay was carried out to make sure the effect of DKK1 in BMP9-induced Osteogenesis differentiation of MSCs in vivo. And the expression of DKK1 induced by BMP9 was analyzed through q PCR.Results: Dkk1 significantly decreased not only BMP9-induced Alkaline phosphatase(ALP) activity, but also the expression of osteocalcin(OC) and osteopontin(OPN) and matrix mineralization of C3H10T1/2 cells. Furthermore, Dkk1 overexpression inhibited BMP9-induced ectopic bone formation of MSCs in vivo. Meanwhile,overexpression of BMP9 was shown to effectively induce Dkk1 expression in a dose-dependent manner, which was partly blocked by P38 inhibitor SB203580 but not ERK inhibitor PD98059.Conclusions: Wnt signal inhibitors DKK1 could significantly inhibit BMP9-induced osteogenesis differentiation of mesenchymal stem cells both in vivo and in vitro.Meanwhile, the expression of DKK1 indcued by BMP9 may partly via MAPK-P38 pathway. Our findings indicate that Dkk1 may negatively regulate BMP9–induced osteogenic differentiation of MSCs.Part three: To Investigate the Mechanism of DKK1 onBMP9-induced Osteogenic Differentiation of MSCsand the Role of Classic Wnt/β-catenin SignalObjectives: To investigate the potential mechanism of DKK1 on BMP9-induced ostoegenic differentiation of MSCs and role of classic Wnt/β-catenin signal in it.Methods: C3H10T1/2 cells were infected by Ad-BMP9、Ad-DKK1and/or Ad-β-catenin.To measure osteogenic differentiation, the ALP was detected by ALP activity assay, calcium deposition was determined by Alizarin Red S staining. Meanwhile, BMP Smad-responsive luciferase reporter assay was carried out to make sure the transcription levels of Smad,the Protein levels of Smad were detected by Western blot. The expression of Runx2 was detected by q PCR and Western blot. Combined infection of Ad-β-catenin, ALP activity,the Smad expression and the calcium deposition were carried out to make sure if DKK1 inhibited BMP9-induced Osteogenic Differentiation of MSCs by classic Wnt/β-catenin signal.Results: Dkk1 significantly decreased not only BMP9-induced Smad activity, but also the expression of Runx2 of C3H10T1/2 cells byBMP-Smad pathway. Furthermore, Dkk1 inhibited BMP9-induced expression of Wnt/β-catenin signal. Meanwhile, inhibition of β-catenin expression also diminished BMP9-induced osteogenic differentiation of MSCs. Overexpression of β-catenin was shown to effectively rescue the inhibition of Dkk1 in BMP9-induced osteogenic differentiation of MSCs.Conclusions: DKK1 could significantly inhibit BMP9-induced osteogenesis differentiation of MSCs by BMP-Smad-Runx2 axis.Meanwhile, canonical Wnt signaling may acts synergistically with BMP9 in osteogenic differentiation. And DKK1 may inhibit BMP9-induced osteogenic differentiation of MSCs by canonical Wnt /β-catenin signal.