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

一些研究表明，与在塑料基质上形成相比，当破骨细胞祖细胞在骨基质上孵育时，成熟破骨细胞的形成显著增加。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性作用。本研究在本次科研资助项目中，我们从牛骨中分离纯化了骨细胞成熟诱导因子，并获得了以下结果。(1)用1 α，25-（OH）_2D_3诱导的抗酒石酸酸性磷酸酶（TRAP）阳性细胞与牛骨EDTA提取物联合培养，可吸收牙本质切片。(2)在用EDTA脱矿期间从牛骨粉中提取OMIFs，并通过Superdex 75制备级凝胶过滤分级和纯化。OMIFs活性的峰作为约12 KD的物质从凝胶过滤柱洗脱。(2)具有OMIFs活性的峰级分在约60 mM磷酸钠（pH 6.8）处通过羟基磷灰石柱洗脱为主峰。(3)通过用0.02M Bis-Tris丙烷（pH7.0）平衡的Mono Q，在约250 mM NaCl下将OMIF洗脱为主峰。（4）用羟基磷灰石柱进行反相高效液相色谱分离，得到了多个峰。高分辨16.5%聚丙烯酰胺凝胶电泳结果表明，OMIFs的分子量分别为5.1KD、6.8KD和8.4KD。

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.