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骨形态发生蛋白（BMP），用高效液相色谱法进行凝胶过滤。刺激DNA合成，抑制它，刺激碱性磷酸酶活性的牙髓细胞培养的馏分可以分离，这表明生长因子和牙本质诱导因子可能是不相同的。已知胰岛素样生长因子I、II存在于牙本质中，刺激牙髓细胞生长。因此，我们尝试分离牙本质诱导因子如下。将0.15-0.50M NaCl肝素琼脂糖亲和层析流出物植入大鼠皮下。诱导大量骨形成，骨髓碱性磷酸酶活性高。在第14天，在牙髓细胞培养物中，流出物部分使用磷酸蛋白抗体以剂量依赖性方式增加免疫荧光染色。北方印迹分析显示，与其他组分相比，流出物组分也增加了骨钙素mRNA的表达。肝素琼脂糖亲和层析法可有效地从BMP粗品中分离牙本质诱导因子。我们正在开发更有效和更方便的检测方法。

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.