Purification and identification of factors involved in osteoclastic maturation from bovine bone matrix

牛骨基质中破骨细胞成熟相关因子的纯化及鉴定

基本信息

批准号：
05671513
负责人：
AMANO Shigeru
金额：
$ 1.15万
依托单位：
Meikai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05671513/
关键词：
Osteoclast Differentiation Bone matrix

项目摘要

Several studies have shown that formation of mature osteoclasts significantly increases when osteoclast progenitors are incubated on bone matrix compared with the formation on a plastic substratum. These observations have suggested that some components of bone matrix play a functional role in formation of mature osteoclasts. However, detailed characterization of these components derived from bone matrix has not yet been made.In the present grant-aid for scientific research, we purified the osteoclastic maturation-inducing factors from bovine bone, and obtained the following results.(1) Although tartrate-resistant acid phosphatase (TRAP) -positive cells formed from calvarial cells in the presence of 1alpha, 25- (OH) _2D_3 on plastic culture dishes could not resorb dentine slices, 1alpha, 25- (OH) _2D_3-induced TRAP-positive cells formed in combination with EDTA-extracts from bovine bone could resorb it.(2) The OMIFs were extracted from bovine bone powder during demineralization with EDTA,and fractionated and purified by gel filtration over Superdex 75 prep grade. The peak of OMIFs activity was eluted as an approximately 12KD species from the gel filtration column.(2) The peak fraction having OMIFs activity eluted as a major peak at about 60mM sodium phosphate (pH6.8) by the hydroxyapatite column.(3) The OMIFs eluted as a major peak at about 250mM NaCl by Mono Q equilibrated with 0.02M Bis-Tris propane (pH7.0).(4)The OMIFs were separated into several peaks by reversed-phase HPLC.the hydroxyapatite column. The molecular weights of OMIFs estimated from high-resolution 16.5% polyacrylamide gel electrophoresis were 5.1KD,6.8KD and 8.4KD respectively.In further studies, we will analyze the amino-acid sequence and define physicochemical characteristics of the purified OMIFs.

多项研究表明，与在塑料基质上形成的破骨细胞祖细胞相比，当破骨细胞祖细胞在骨基质上培养时，成熟破骨细胞的形成显着增加。这些观察结果表明骨基质的某些成分在成熟破骨细胞的形成中发挥功能作用。然而，这些来自骨基质的成分尚未得到详细表征。在目前的科研资助中，我们从牛骨中纯化了破骨细胞成熟诱导因子，并得到了以下结果。（1）虽然酒石酸盐- 抗性酸性磷酸酶 (TRAP) - 在塑料培养皿上存在 1α, 25- (OH) _2D_3 的情况下由颅骨细胞形成的阳性细胞不能吸收牙本质切片，1α， 25-(OH)_2D_3诱导的TRAP阳性细胞与牛骨EDTA提取物结合形成的TRAP阳性细胞可以吸收它。(2)在EDTA脱矿过程中从牛骨粉中提取OMIFs，并通过凝胶过滤进行分级和纯化。 Superdex 75 预备级。 OMIF活性的峰作为约12KD物质从凝胶过滤柱洗脱。(2)具有OMIF活性的峰级分通过羟基磷灰石柱在约60mM磷酸钠(pH6.8)处作为主峰洗脱。(3)通过用 0.02M Bis-Tris 丙烷平衡的 Mono Q，OMIF 在约 250mM NaCl 处作为主峰洗脱(pH7.0)。(4)反相HPLC.羟基磷灰石柱将OMIFs分离成多个峰。通过高分辨率16.5%聚丙烯酰胺凝胶电泳估计OMIF的分子量分别为5.1KD、6.8KD和8.4KD。在进一步的研究中，我们将分析纯化的OMIF的氨基酸序列并确定其理化特性。