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

一些研究表明，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比在塑料基质上的形成明显增加。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起功能作用。然而，这些来源于骨基质的成分的详细表征尚未完成。在本次科研资助中，我们从牛骨中纯化了破骨细胞成熟诱导因子，获得了以下结果：(1) 1 α, 25- (OH) _2D_3诱导的TRAP阳性细胞与牛骨edta提取物联合形成的TRAP阳性细胞能吸收牙本质片，而在塑料培养盘上由颅骨细胞形成的抗酒石酸酸性磷酸酶（TRAP）阳性细胞不能吸收牙本质片。(2) EDTA脱矿提取牛骨粉中的omif，经Superdex 75预处理级凝胶过滤分离纯化。凝胶过滤柱洗脱出的OMIFs活性峰约为12KD。(2)在约60mM磷酸钠（pH6.8）处，羟基磷灰石柱洗脱的OMIFs活性峰为主峰。(3)用0.02M Bis-Tris丙烷（pH7.0）平衡的Mono Q在约250mM NaCl下洗脱的OMIFs为主峰。(4)反相高效液相色谱法将omif分离成多个峰。羟基磷灰石柱。高分辨率16.5%聚丙烯酰胺凝胶电泳测得的OMIFs分子量分别为5.1KD、6.8KD和8.4KD。在进一步的研究中，我们将分析氨基酸序列并定义纯化的OMIFs的物理化学特性。

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.