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

从牛脱矿牙本质中提取粗骨形成蛋白(BMP)，用高效液相色谱法对其进行凝胶过滤。可分离出促进DNA合成、抑制DNA合成和刺激牙髓细胞碱性磷酸酶活性的组分，提示生长因子和牙本质诱导因子可能不同。已知牙本质中含有的胰岛素样生长因子I、II促进牙髓细胞的生长。因此，我们尝试分离牙本质诱导因子如下。将0.15-0.50M氯化钠肝素琼脂糖亲和层析的流出物部分植入大鼠皮下。诱导出大量碱性磷酸酶活性较高的骨髓组织。在第14天的牙髓细胞培养中，流出组分以剂量依赖的方式增加了使用磷酸磷脂抗体的免疫荧光染色。Northern印迹分析表明，与其他组分相比，流出组分也能促进骨钙素mRNA的表达。肝素琼脂糖亲和层析可能是从粗制BMP中分离牙本质诱导因子的有效方法。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.