Preparation Of Osteogenic Growth Peptide Antibody And Its Mitogenic Mechanism On NIH3T3 Cells

Objective: Osteogenic growth peptide (OGP), a small molecular polypeptide with 14 amino acid , is purified from the conditioned medium recovered from cultured regenerating rat bone marrow. In vivo OGP is physiologically present in the serum and is completely homologous in most mammal species. It has been showed that OGP and the carboxy-teminal fragment of OGP promote bone formation and hemopoiesis . In vitro it was showed that OGP can regulate proliferation in osteogenic MC3T3E1 cell, NIH3T3 fibroblast, osteogenic Rosl7/2 cell and other cells. But now the mechanism of the effects on cells by OGP is not clear. The present experiment was to using designed poly-OGP to prepare OGP antibody. Survey the mitogenic action of sOGP on NIH3T3 cells. Immunoassays by OGP antibody, and other techniques were used to study the mitogenic mechanisms on NIH3T3 cells by OGP.Methods: In the first part of the experiments, Merrifield solid phase peptide methods were applied for the peptide synthesis. Boc system was performed for Osteogenic growth peptide (OGP). HPLC, mass spectrometric analysis demonstrate the molecular accuracy and precision. Poly-OGP and KLH-OGP were used as antigen to immunize New Zealand rabbits for the preparation of antiserum. Using the antiserum, we measured the content of the sOGP with RIA method or competitive ELISA methodIn the second part of the experiments, the fibroblastic NIH3T3 cells were seeded in the 10% NCS-1640 at 5 × 10⁴/ml in 96-well-tissue culture plate. Different concentrations of peptide [sOGP or sOGP(10-14)] were added to the cultures. MTT method were performed after 48h. At 10^-11mol/l M peptide concentration, cell counts and cell cycle testing by Flow Cytometry(FCM) were carried out after 48h to evaluate the proliferation. RT-PCR and ELISA were performed to evaluate the changes of type I collagen.In the third part of the experiments, the changes of OGP on NIH3T3 cell membrane were surveyed by FCM FITC with OGP antibody. After adding the OGP antiserum to the culture, we surveyed the changes of cell proliferation. And we also...