The Mechanism Of Osteogenic Growth Peptide On The Therapeutic Effect Of Osteoprotegerin Deficient Mice

Objective:Osteoporosis is a frequent disease due to its prevalence. With China entering the aging, the incidence of osteoporotic fracture increases year by year and it's a major disabled factor for the elderly. The osteoprotegerin (OPG) deficient mouse exhibits an osteoporotic phenotype and could be useful for screening and evaluation of drugs used in treatment of osteoporosis. Osteogenic growth peptide (OGP), is a naturally occurring tetradecapeptide and in vitro studies, which were performed on cellular systems based on osteoblastic-like cell lines or mouse stromal cells, having demonstrated that OGP increased osteoblast proliferation, alkaline phosphatase (ALP) activity and mineralization. To illuminate the effect of OGP on boen mesenchymal stem cells (BMSCs) derived from OPG deficient mice proliferation and differentiation, we compare it with bone morphogenetic protein-2(BMP-2), and provide theoretical evidences for treating osteoporosis using the OGP.Methods:1. BMSCs culture:OPG deficient mice (male,12 weeks old, weighing about 25g) were bought from Shanghai Research Center for Biomodel Organisms. The BMSCs were cultured with Dulbecco's-modified Eagle's Medium-Low Glucose (DMEM-LG, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Sigma, USA),100μg/mL streptomycin, and 100 U/mL penicillin,and 10-8 mol/L dexamethasone,10 mmol/Lβ-glycerophosphate and 50μg/ml ascorbic acid, here after termed experimental culture medium. We observed the morphological changes of BMSCs derived from OPG deficient mice.2. Proliferation and ALP assay:To assess the effects of OGP on the proliferation and ALP assay of BMSCs derived from OPG deficient mice, we divided the passage first(P1) cells into OGP groups,non-treated groups and BMP-2 groups and observed 1 d,3 d,5 d,7 d four times. We used methyl thiazolyl tetrazolium (MTT) andρ-nitrophenylphosphate (PNPP) method to measure proliferation and ALP assay respectively. 3. Quantitative real-time PCR:P1 cells were cultured in experimental culture medium for 3 days and were divided into 3 groups and extracted total RNA. We quantified mRNA of Runt-related transcription factore 2 (Runx2),Osterix,CDK2,Cyclin A and detected the mRNA level changes in these three groups.4. Western-blot analysis:P1 cells were cultured in experimental culture medium for 7 days and were divided into 3 groups. We observed the levels of the protein which were related with the proliferation and differentiation and analyzed the possible mechanism.Results:1. The BMSCs derived from OPG deficient mice grew slowly and difficultly reached confluency in passage third. So we used the P1. However there were no significant changes in morphology.2. OGP promoted the proliferation of BMSCs (derived from OPG deficient mice) significantly than that of the other two groups(P<0.05) when cells were cultured in experimental culture medium on the third,fifth and seventh days.3. OGP increased mRNA level of CDK2 and CyclinA, two key cell cycle regulators (P<0.01). The mRNA level of Runx2 and Osterix in OGP-treated cells was similar to non-treated cells, and inferior to BMP-2 treated-cells.4. All antibodies were found on western blots. OGP increased protein levels of CDK2 and CyclinA than that of the other two groups (P<0.01); and protein levels of Runx2 and Osterix in BMP-2 groups showed an additional elevation over the other groups (P<0.01), but the OGP groups were similar to that of non-treated groups.Conclusions:1. OGP enhances proliferation of BMSCs (derived from OPG deficient mice) which were cultured in experimental culture medium, however, its effect on ALP expression was inferior to that of BMP-2.2. OGP enhances the proliferation of BMSCs from OPG deficient mice by CDK2/Cyclin A pathway, which promotes S phase of the cell cycle.3. OGP is inferior to BMP-2 in terms of BMSCs differentiation. We presume that the absence of OPG might cut down the effect. Further studies looking at these mechanisms are needed.