Regulate Of The Adipogenic And Osteogenic Differentiation Of BMSCs By Sh-RNA-DKK1

[Objective] By the interference of DKK1, the regulating effects of DKK1on early adipogenic and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) were discussed and its mechanism was analyzed.[Methods]1. In the early adipogenic and osteogenic induction culture of BMSCs, the gene expression level of DKK1was detected by Real time-PCR;The gene expression of DKK1of BMSCs acted by hormone was tested.2. According to rules of shRNA target sites selection,3DKK1shRNA sequences were chosen from shRNA database and Ambion shRNA database.The optimal interfering sequence was screened by construction of DKK1over-expression vector and identified by restriction analysis, PCR and sequencing via construction of homologus recombination DKK1-shRNA adenovirus vector in vitro.3. BMSCs were transfected by rAd-EGFP and its fluorescence expression was observed. The effects of adenovirus on cell proliferation were analyzed. After transfected by Ad-DKK1-shRNA, BMSCs were induced into adipose and bone. Specific differentiation factors and its expression levels of related protein were detected. Then the difference between the experimental group and the control was analyzed. The morphologic change was observed after tissue staining.[Results]1. The relative gene expression level of DKK1was tested by real time-PCR in the adipogenic and osteogenic differentiation of BMSCs of rats within48hours,then obtained the Ct value by the objective gene amplification kinetics curves and caculated the relative gene expression of ΔCt value by theβ-actin correction,and used two independent sample t-test to compare the groups:adipogenic group and the normal control group, When the adipogenic differentiation for6,12,24, and48hours, the gene expression level of DKK1is higher than that in the normal group. The difference was statistically significant, P<0.01.Osteogenic group and the normal control group, when osteogenic differentiation for0.5,6hours, two time points,Dkkl gene expression was significantly lower than the control group.The difference was statistically significant, P<0.05.When hormone acted on BMSCs for3,6,12,24hours respectively,DKK1gene expression was tested by real time-pcr. The result showed that DKK1expression of BMSCs was increased significantly under the action of hormone.2. Expression vectors including pYr-1.1-DKK1-shRNA-153、pYr-1.1-DKK1-shRNA-155、 pYr-1.1-DKK1-shRNA-Am have been successfully constructed. DKK1has been detected via RT-PCR and Western blot assays respectively form above3vectors.It indicated that pYr-1.1-DKK1-shRNA-153is the optimal interfering sequence.3. By fluorescence observation of transfected BMSCs, the groups of MOI-10,20obtained higher transfection rate. The groups of MOI-5,10,20,30,40,50pfu/cell did not have obvious effect on cell proliferation of BMSCs after adenovirus tranfection. sh-DKK1had significant inhibition effects on adipogenic differentiation. Sh-DKK1could improve the expression levels of osteogenic protein, Osteocalcin and coll-Ⅰ.[Conclusion] Dkkl might play an important role in regulation of the early adipogenic and osteogenic diffrerntiation of bone marrow mesenchymal stem cells(BMSCs). The hormone might regulate DKK1expression to play an role in regulation of adipogenic and osteogenic differentiation.DKK1interference had significant inhibition effects on early adipogenic differentiation of BMSCs. It could improve the expression levels of osteogenesis protein and promote early osteogenesis differentiation. DKK1played an important role in the regulation of early adipogenic and osteogenic differentiation of BMSCs.