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

牙本质和牙髓中牙髓细胞生长因子（PGF）的分离及其在断牙中的应用

• 批准号: 02454438
• 负责人: NAGASAWA Hisashi
• 金额: $ 4.35万
• 依托单位: Kyushu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-02454438/
• 关键词: pulp cell; odontoblast; growth factor; differentiation factor; phosphophoryn; osteocalcin; pulpotomy; Bone Morphogenetic Protein; 骨形成因子; 歯髄培養細胞; DNA合成; アルカリフォスファタ-ゼ; 細胞増殖; 細胞分化; 蛍光抗体法; III型コラ-ゲン; 歯髄; 象牙質; 初代培養; 高速液体クロマトグラフィ-

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.

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。

项目成果

M.Nakashima: "The effects of growth factors on DNA synthesis,proteoglycan synthesis and alkaline phosphatase activity in bovine dental pulp cells" Archs oral Biol.

M.Nakashima：“生长因子对牛牙髓细胞 DNA 合成、蛋白聚糖合成和碱性磷酸酶活性的影响”Archsoral Biol。

Misako Nakashima: "Mitogenic and dentine-inductive effects of crude bone morphogenetic protein from bone and dentine in primary adult pulp cell culture" Oral Surg Oral Med Oral Pathol. 73. 484-9 (1992)

Misako Nakashima：“原代成人牙髓细胞培养物中来自骨和牙本质的粗骨形态发生蛋白的有丝分裂和牙本质诱导作用”口腔外科口腔医学口腔病理学。

Misako Nakashima: "Mitogenic and dentine-inductive effects of crude bone morphogenetic protein from bone and dentine in primary adult pulp cell culture" Oral Surg Oral Med Oral Pathol. 73. 484-489 (1992)

Misako Nakashima：“原代成人牙髓细胞培养物中来自骨和牙本质的粗骨形态发生蛋白的有丝分裂和牙本质诱导作用”口腔外科口腔医学口腔病理学。

M.Nakashima: "Mitogeneic and dentinーinductive effects of crude bone morphogenetic protein from bone and dentin in primary adult pulp cell culture" Oral Surg Oral Med Oral Pathol.

M.Nakashima：“原代成体牙髓细胞培养物中来自骨和牙本质的粗制骨形态发生蛋白的有丝分裂和牙本质诱导作用”口腔外科口腔医学口腔病理学。