The Study About OGP And The Mechanism Of OGP To Promote Rat Bone Marrow Mesenchymal Stem Cells To Osteogenetic Differentiation

The Osteogenic Growth Peptide(OGP)is a well-defined.14-amino-acid peptide which is coded by histone H4 gene.OGP represents an interesting case of a "housekeeping" peptide that plays an important role in osteogenesis and hematopoiesis. OGP has attracted considerable clinical interest as bone anabolic agents and hematopoietic stimulators.OGP increases bone formation and trabecular bone density and stimulates fracture healing when administered to mice and rats.In cultures of osteoblastic and other bone marrow stromal cells,derived from human and other mammalian species,OGP regulates proliferation,alkaline phosphatase activity and matrix mineralization via an autocrine/paracrine mechanism.OGP can enhance hematopoiesis,including the stimulation of bone marrow transplant engraftment and hematopoietic regeneration after ablative chemotherapy.Apparently,the hematopoietic effect of OGP is secondary to its effect on the bone marrow stroma.Detailed structure-activity relationship study identified the side chains of Tyr10 and Phe12 as the principal pharmacophores for OGP-like activity. We hypothesized that the activity of osteogenesis and hematopoiesis of OGP may positively regulate bone formation by directly affecting Bone Marrow Mesenchymal Stem Cells(BMSCs).Bone Marrow Mesenchymal Stem Cells are multipotent cells capable of differentiating to many lineages like osteogenic and adipogenic lineages when stimulated under appropriate conditions.Bone Marrow Mesenchymal Stem Cells constitute the bone marrow Micro-environment which is necessary for proliferation and differentiation of hematopoietic cells.We are interested in the relationship between the activity of osteogenesis and hematopoiesis of OGP and Bone Marrow Mesenchymal Stem Cells.We emphasized in the following aspects:(1)We examined the effect of OGP on the proliferation and differentiation of the cultured rat Bone Marrow Mesenchymal Stem Cells(rBMSCs).The results indicated that OGP at the concentration of 10^-10mol/L and 10^-11mol/L promoted the proliferation of rat Bone Marrow Stromal Cells,while at the concentrations of 10^-8mol/L and 10^-9mol/L advanced rat Bone Marrow Stromal Cells differentiating to osteogenesis.The RT-PCR result and the following DNA chip result of the transcription factor Cbfα1 and Osteocalcin indicated that the effect of OGP to promote rBMSCs osteogenetic differentiation may be not related with the transcription factor Cbfα1 and Osteogenic Growth Peptide induced rBMSCs had not completely differentiated to osteogenetic cells.2)The study of the expression profile change of Osteogenic Growth Peptide induced rBMSCs with 27K Rat Genome Array.The result of DNA chip indicated that compared with the control,a total of 145 genes were found to be expressed differently in the presence of OGP,including 91 down-regulated and 54 up-regulated.Among them,35 genes were related with bone fracture healing,including 4 genes related with angiogenesis, 9 gens related with osteogenesis,8 genes related with cell proliferation and differentiation, and 14 genes related with inflammatory and immune response.The result layed foundation for studying the molecular mechanism of OGP induced rBMSCs to osteogenetic differentiation.(3)The comparison of the activity of osteogenetic differentiation between rBMSCs transfected with recombined vectors and Osteogenic Growth Peptide induced rBMSCs. We constructed recombinant OGP sense vectors and Pre-OGP anti-sense vectors based on pEGFP-N1 vector and successfully transfected into rBMSCs.The observation of fluorescence showed that recombinant OGP sense vectors and Pre-OGP anti-sense vectors could successfully transfect rBMSCs.The detection of the Alkaline phosphatase activaty showed that Pre-OGP anti-sense vectors transfected rBMSCs were restrained to osteogenetic differentiation,and OGP sense vectors transfected rBMSCs were promoted to osteogenetic differentiation like Osteogenic Growth Peptide induced rBMSCs. (4)Establishment of enzyme immunoassay methods for OGP.We modified the Enzyme linked immununosorbent assay for OGP.which can be used for the detection of the serum OGP in clinic.The sensitivity of detection was greatly improved.