The mechanism of bone formation with reference t the phosphoprotein in the bone matrix

用超微结构、免疫组织化学和生化方法研究酸性磷酸蛋白与骨髓基质钙化的关系。超微结构分析显示，小颗粒(~0.5μm)分布于类骨质的深部，其分布规律与钙化颗粒相似。染色后全部染成蓝色的区域与基质内钙化区域的分布规律相似。经全染蓝染色的基质成分生化分析表明，该基质成分与鸡骨桥蛋白相似。此外，随着钙化程度的加深，骨桥蛋白mRNA在髓质中的表达增强。分别针对从骨髓骨矿物相获得的蛋白质组分和以部分氨基酸序列为基础的合成肽，制备多克隆抗体。用该蛋白产生的抗体在基质和骨细胞中呈阳性反应，而用该肽产生的抗体仅在破骨细胞和成骨细胞中呈阳性反应。这些结果表明骨桥蛋白在髓质骨基质中大量存在，并随着基质钙化的进行而增加，提示骨桥蛋白与髓质骨钙化有关。

The relationship between acidic phosphoprotein and the calcification of medullary bone matrix was examined employing ultrastructural, immunohistochemical and biochemical methods. Ultrastructural analysis showed that small globules (〜0.5 μm) were distributed at the deeper parts of osteoid, and the distribution pattern of the globules was similar to that of calcifying globules. The distribution pattern of the areas stained blue by stains-all staining was also similar to that of calcified areas in the matrix. Biochemical analyses of the matrix component stained blue by stains-all staining showed the similarity to chicken osteopontin. Furthermore, according as calcification progressed, the expression of quail osteopontin mRNA was enhanced in medullary bone. Polyclonal antibodies were raised against the protein fraction obtained from mineral phase of medullary bone and the synthetic peptide based the partial sequence of amino acid of quail osteopontin, respectively. Immunohistochemical reaction was positive in the matrix and bone cells with use of the antibody raised from the protein, but the positive reaction was confined to osteoclasts and osteoblasts, using the antibody raised against the peptide. These results indicate that osteopontin abundantly exists in medullary bone matrix and increases according as the matrix calcification advances, and suggest that osteopontin relates to medullary bone calcification.