Study On Expression Of RhBMP-7 And Its Biological Effect On Human Periodontal Ligament Fibroblasts

Objective:To express human bone morphogenetic proteins 7(rhBMP-7) by Genetic Enginerrting Technique, and to evaluate the biological effects of rhBMP-7 on the proliferation and differentiation of primary culture of human periodontal ligament fibroblasts (hPDLFs). Methods: using pET-3c as vector , the cloned rhBMP-7 by SOE-PCR technique was expressed in E.coli BL21(DE3)pLysS after IPTG induction using Modified traditional tissue method to obtained primary culture of hPDLFs. After being treated with different concentration of rhBMP-7, the proliferation of hPDLFs was evaluated via Flow Cytomery (FCM) analysis colorimetirc assay; For the evaluation of osteogenic differentiation, the alkaline phosphatase(ALP) activity was determined, ALP staining used by adoping Calcium-cobalt, calcification nod staining used by Alizarin Red S staining method etc.Ten marker gene/protein in osteogenic cells, such as ALP, OC, Collagen I , BSP, OPG, OPN, Runx2, Osx, Dlx5, Msx2, were also detecked with RT-PCR technique. Results: Highly pure rhBMP-7 (100ug/mL) was obtained. Primary culture of hPDLFs was successful, a nd after 10^th passage,proliferation index (PrI) of hPDLFs was obviously declined and number of cells was reduced. The treatment with rhBMP-7 increased the ALP activity, staining positive rate. Alizarin Red S staining of cells indicated that rhBMP-7 enhanced calcium mineral deposition and the mineralization percent was 10 times more than that of the control. The effective dose of rhBMP-7...