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Preparation of Osteogenic Growth Peptide Antibody and Its Mitogenic Mechanism on NIH3T3 Cells

Author: WangXiaoFeng
Tutor: ChenZhongWei；ChenTongYi；CuiDaFu；ShiDeYuan；JiangXiaoXing
School: Fudan University
Course: Surgery
Keywords: Osteogenic growth peptide (OGP) NIH3T3 Fibroblast Proliferation activity Flow cytometry determination (FCM) Pertussis toxin (PTX)
CLC: R392
Type: PhD thesis
Year: 2005
Downloads: 102
Quote: 0
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Abstract


Objective: osteogenic growth peptide (OGP) is a purified a 14 amino acids of the small molecule peptide isolated from bone marrow cultured stromal regeneration after injury. OGP widely present in mammalian serum, evolutionarily conserved, it has good homology. The study found the OGP its C-terminal 5 peptide can promote osteoblasts and hematopoiesis. In the osteoblasts the in vitro MC3T3E1 into, NIH3T3 fibroblasts and osteoblasts Rosl7 / 2 cells and promote the proliferation and differentiation have. But OGP cell mechanism of action is not clear. To this end, the present study, the Chinese Academy of Sciences Biochemical synthetic sOGP of design poly-OGP OGP antibody was prepared to observe the sOGP effect on the proliferation of NIH3T3 cells, and prepared antibody immunoassay and other technologies to explore the OGP its C terminal 5 peptide NIH3T3 cell proliferation mechanism. Method: The experiment was first portion Merrifield solid phase synthesis method oligopeptide. Synthesis OGP with Boc system. HPLC, Mass Spectrometry analysis of the uniformity and accuracy of the identification of its molecules. Preparation of poly branch peptide (Poly-OGP) and KLH-OGP. Poly-OGP and KLH-OGP as an antigen to immunize New Zealand rabbit antiserum. Antiserum by radioimmunoassay (RIA) and ELISA determination of sOGP concentration. Experiment second portion of NIH3T3 cells to cell concentration of 5 × 10 4 / ml, were inoculated into 96-well culture plate, using 10% NCS-1640 culture medium, containing different concentrations the sOGP or sOGP (10-14). 48 hours later, cell proliferation was observed by MTT assay. Cell concentration of 5 × 10 4 / ml in the cell culture medium of sOGP or sOGP (10-14) the concentration is adjusted to 10 -11 mol / l, 48 hours after the observation of cell proliferation, cell counting, cell cycle detection observed changes in type I collagen by RT-PCR and ELISA. The third part of the experiment using flow cytometry FITC fluorescence staining, observed in NIH3T3 cell surface proliferation the concentration sOGP and sOGP (10-14) under the action of OGP change. Observed in OGP antiserum, sOGP sOGP (10-14) mitogenic effect change. Observed in pertussis toxin (PTX), a Gi inhibitor effect, OGP and OGP (10-14), promoting cell proliferation, membrane OGP immunofluorescence staining and cell cycle changes. Results: synthetic OGP high degree of uniformity, purity by HPLC identification of molecular 99.28%, by mass spectrometry analysis sOGP molecular weight of 1523.5, 1523.75 consistent with the theoretical molecular weight. The average molecular weight of Poly-OGP is the 40KD about. Poly-OGP can be immunized animal antisera obtained can be measured. MTT assay, cell cycle, cell counting method can observe the the different concentration sOGP and sOGP (10-14)

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