Separation of Pulp Cell Growth Factor(PGF) in dentine and pulp and application on amputated pulp

采用高效液相色谱法对从牛脱矿牙本质中提取的骨形态发生蛋白进行了凝胶过滤。在培养的牙髓细胞上，刺激DNA合成、抑制DNA合成和刺激碱性磷酸酶活性的部分可以分离出来，说明生长因子和牙本质诱导因子可能不完全相同。已知牙本质中含有的胰岛素样生长因子I、II刺激牙髓细胞生长。因此，我们试图通过以下方法分离牙本质诱导因子。采用肝素- sepharose亲和层析法，取0.15 ~ 0.5 m NaCl浸出液皮下植入大鼠。培养出大量碱性磷酸酶活性高的骨髓。在牙髓细胞培养的第14天，用phosphophoryn抗体对流出液进行剂量依赖性免疫荧光染色。Northern blot分析显示，与其他馏分相比，出水馏分也增加了骨钙素mRNA的表达。肝素- sepharose亲和层析法可有效分离粗BMP中牙本质诱导因子。目前，我们正在开发更有效、更方便的检测方法。

Crude bone morphogenetic protein (BMP) extracted from bovine demineralized dentine was gel filtrated by high performance liquid chromatography. The fractions which stimulated DNA synthesis, inhibited it and stimulated alkaline phosphatase activity on pulp cells in culture could be separated, suggesting that the growth factor and dentine inducible factor may be not identical. Insulin-like growth factor I, II which has known contained in dentine stimulated pulp cell growth. We, therefore, tried to isolate dentine inducible factor as follows. An effluent fraction by 0.15-0.50M NaCl in heparin sepharose affinity chromatography was implanted subcutaneously in rat. Large amount of bone with bone marrow which was high in alkaline phosphatase activity was induced. The effluent fraction increased dose-dependently immunofluorescent staining using phosphophoryn antibody on day 14 in pulp cell culture. Northern blot analysis revealed that the effluent fraction also increased the expression of osteocalcin mRNA compared with other fractions.Heparin sepharose affinity chromatography might be effective to separate dentine inducible factor from crude BMP. We are now under way to develope more effective and convenient assay method.