Osteogenic Growth Peptide Promotes Pro Liferation And Osteogenic Differentiation Of Mouse Bone Marrow Mesenchymal Stem Cells

ObjectiveMouse bone marrow mesenchymal stem cells(mBMSCs)were isolated by mouse bone slice method.To detect the induction of osteogenic growth peptide 10-14(OGP(10-14))intervention on osteogenic differentiation of mBMSCs and its effect on cell cycle cyclin B1/CDK2 pathway of cell proliferation.This study is expected to provide a basis for the clinical treatment of bone injury by OGP(10-14).MethodsExperiment was divided into control group and osteogenesis growth peptide group(OGP(10-14)group).Control group was given osteogenesis induction medium,containing 0.1 μmol/L dexamethasone,10mmol/L β-glycerol phosphate,50μmol/L vitamin C,0.1μmol/L glutamine,10%FBS and 1%antibiotic),changed the solution once every 2 or 3 days and induced 14 days.OGP(10-14)group was given osteogenesis induction medium(same as the control group)and 10-5 mol/L OGP(10-14),changed the solution once every 2 or 3 days and induced 14 days.MTS method was used to detect the proliferation of mBMSCs induced at 24h,48h,and 72h.The level of osteogenic differentiation of mBMSCs was detected on 7d and 14d by Alizarin red staining.qPCR and Western blot were used to detect mRNA levels and protein expressions of important osteogenic factors of β-catenin,RUNX2,BSP,and cycle-related factors of cyclin B1,CDK2,c-Myc.Results1.The morphology of the third-generation cells of primary mBMSCs culture were uniform cell morphology,long spindle shape and whirlpool fusion.Flow cytometry analysis of surface marker molecules showed that mBMSCs expressed high levels of CD29(90.3±7.5)and CD90(82.5±8.4),and low levels of CD45(3.6±0.4)and CD11b/c(5.8±0.9).2.MTS results showed that OGP(10-14)significantly promoted the proliferation of mBMSCs,and the proliferation levels were significantly higher than that of the control group at 24h,48h and 72h(t=3.505,P=0.039;t=6.940,P=0.006,t=6.803,P=0.007)。3.The results of alizarin red staining showed that mBMSCs of the control group and the OGP(10-14)group differentiated into osteoblasts under the induction medium,with some cells growing in clusters and colonies.With the extension of induction time,red layered mineralized nodules appeared.Mineralized nodules of mBMSCs in the OGP(10-14)group increased compared with the normal group,suggesting that OGP(10-14)promoted osteogenic differentiation of mBMSCs.4.The results of qPCR and Western blot showed that OGP(10-14)increased the mRNA levels of β-catenin,RUNX2,and BSP(t=6.224,P=0.008;t=4.985,P=0.016;t=5.560,P=0.012),and promoted protein expression compared with the control group(t=7.053,P=0.006;t=8.927,P=0.003;t=8.580,P=0.003).Compared with the control group,the mRNA levels of cyclin B1,CDK2 and c-Myc in the OGP(10-14)group were significantly increased(t=8.917,P=0.003;t=6.407,P=0.008;t=6.196,P=0.009),and the protein expressions were increased to varying degrees(t=10.821,P=0.000;t=8.200,P=0.004;t=7.181,P=0.006).ConclusionsOGP(10-14)promoted osteogenic differentiation of BMSCs by Wnt/β-catenin signal,and triggered cell cycle of cyclin B1/CDK2 pathway to promote mBMSCs proliferation,thus increasing bone formation.