Osteoblast Differentiation Induced BMP-2 Plasmid DNA And Crosstalk Between BMP-2 And Wnt/Beta-Catenin Signaling

Background: Marrow stromal cells (MSCs) using with genetic therapy and suitable delivery method are bright prospects -for bone tissue engineering and bone repair. Transferring the bone morpho genetic protein 2 (BMP-2) genes into the tissues or cells have been successfully used for clinical bone repair. Numerous of pathways including BMP signaling and Wnt pathway play an important role for regulation of osteoinduction and proliferation. Becuase of autologous variable mechanism interaction , the cross talk between the BMP pathway and the Wnt/p-catenin pathway contributes toward the sophisticated and complexity during the osteogenic differentiation . A detailed and exact mechanism is currently need to detect for the future targeted and genetic therapy .Objective: This study aim to investigated the cross talk between the BMP2 pathway and the Wnt/β-catenin pathway during the osteogenic differentiation in C3H10T1/2 cells transfected into BMP-2plasmid DNA.By the way, the osteogenic activity and effectivity of hBMP-2plasmid DNA transferring C3HIOT1/2 cells had been researched.Method and Result1 The osteogenesis potency of C3H10T1/2 cells and osteogenic induction effectivity of BMP-2plasmid DNAMethod:First ,we used BMP-2plasmid DNA transfer into C3H10T1/2 cells with a Lipofectamine3000 and p3000 transfected protocol and G418 selection using stable cells. Subsequently,the concentration of medium supernatant liquor BMP-2 was detected by BMP-2 ELISA kit, and multiplication capacity was detected by CCK-8 kit. Finally, the ALP activity was quantified by ALP stain and ALP kit as measures of osteoblast differentiation.Calcium deposition induced by BMP2 was quantified by extracting the Alizarin red stain .Result:(1)BMP-2plasmid DNA can be successfully transfected into C3H10T1/2 cells,which is a safety and effective gene transfer method .(2)The concentration of BMP-2 in transfer group higher than blank group and control plasmid (p <0.05). (3)CCK-8 result show that all group and transfer group achived higher proliferation rate than other groupin day 4 to day 8 and increasing in a time-dependent manner. (4)The quantitative analysis of ALP activity of transfer group obviously appear ALP-positive and a higher level than other group . Alizarin red measure appear positive mineralization nodules.Conclusion:BMP-2plasmid is a kind of effective and safety transfection method . C3H10T1/2 cells is suited to been parasitifer for recombination BMP-2 genetic plasmid and succeed to secrete exogenous. BMP-2 or BMP-2 gene distinctly facilitate MSC multiplication and osteogenic differentiation.2The cross talk between the BMP-2 pathway and the Wnt/β-catenin pathway during the osteogenic differentiationMethod:In this part ,we measured crucial cytokines and signal downstream of classical Wnt signalling in BMP-2plasmid transfer cells.The expression of LEF1, Smurf-2 and β-catenin phosphorylation protein were detected by western-blot, and relevant mRNA include LEF1,Smurf-2, β-catenin , AXIN , RUNX2 and BMP-2 test by quantitative polymerase chain reaction.Result:In the stage of osteoblast differentiation inducted by BMP-2. The expression of LEF1 protein and phosphorylation β-catenin were higher in Transfer group than Blank group , statistical result is t=4.256 ,p=0.013 and t=7.609 , p=0.013, respectively .However, Expression of Smurf-2 protein has a significant different between two group (t=7.609,p=0.013). Statistical analysis was conducted by independent sample t-test.Significant level a=0.05. BMP-2 upregaluted osteogenesis special gene ,RUNX-2, LEF1 and key cytokines in Wnt/β-catenin pathway .At the same time, BMP-2 reduce degradation complexus of β-catenin in cytoplasm.Conclusion:Exploring BMP signaling and Wnt / β-catenin pathway in osteoblast differentiation process crosstalk mechanism, we find (1)BMP-2 up-regulated Wnt / β-catenin pathway key cytokines β-catenin, pβ-catenin, RUNX2, LEF1 expression, involved in activation of Wnt /(3-catenin pathway; (2)BMP signal path through the influence Smurf-2,Axin, GSK3β together to regulate intracellular β-catenin stabilization and savings; (3) Increase in β-catenin phosphorylation levels, and promote activation of β-catenin to the nucleus transduction and downstream signal is transmitted to the network.