Functions of Non-Collagenous Proteins in Dentin

已知牙本质基质中存在多种非胶原成分，如磷蛋白、蛋白多糖、骨结素和血清α-2-HS-糖蛋白。这些成分被认为与牙本质的钙化机制有关。在本研究中，我们打算分离、纯化和鉴定牙本质中的非胶原蛋白，以期阐明它们的生理作用。并且，得到了以下结果。1)加入牙本质磷酸盐后，琼脂体系中的磷酸钙沉淀物数量减少，但有明显的条带形成。去磷酸化的磷酸没什么作用。该蛋白在乳牙和恒牙以及牙冠和根部牙本质中的分布不同。牙本质发育不全的牙本质中不含磷。通过成牙本质细胞培养和免疫印迹鉴定牛磷蛋白的生物合成。2)分析了牙本质中的蛋白多糖，并与骨和软组织中的蛋白多糖进行了比较。阐明了牙本质及相关组织中低分子蛋白多糖核心蛋白的免疫学性质和结构。在软组织中也发现了这种类型的蛋白多糖。3)以成牙本质细胞的mRNA为模板，用抗骨连接蛋白的单抗克隆了牙本质骨连接蛋白基因的碱基序列，确定了该基因的氨基酸序列。该序列与SPARC序列完全相同。用免疫组织化学技术检测该蛋白在不同软组织中的表达。这些结果提示，牙本质中的非胶原成分可能在牙本质形成的发育、分化和钙化过程中发挥调节作用。

Various non-collagenous components such as phosphophoryn, proteoglycans, osteonectin and serum alpha-2-HS-glycoprotein are known to be present in dentin matrix. These components are supposed to be related to the calcification mechanisms in dentin. In the present study we intended to isolate, purify and characterize non-collagenous proteins in dentin for the purpose of elucidation of their physiological roles. And, the following results were obtained. 1) The amount of the precipitates of calcium phosphate in agar gel system decreased when dentin phosphophoryn was added but an effect to produce a sharp band was observed. Dephosphorylated phosphophoryn had no effect. Distribution of this protein was different between deciduous and permanent teeth and between crown and root dentin. Dentinogenesis imperfecta dentin did not contain phosphophoryn. Biosynthesis of bovine phosphophoryn was identified by odontoblast incubation and immunoblotting. 2) Proteoglycans in dentin were analyzed in comparison to those in bone and the soft tissues. Immunological properties and structure of the core protein of low molecular weight proteoglycan in dentin and related tissues were clarified. This type of proteoglycan was also found in the soft tissues. 3) Amino acid sequence of dentin osteonectin was elucidated from base sequence of cDNA prepared by mRNA from odontoblasts and cloned by using monoclonal anti-osteonectin antibody. The sequence was identical to that of SPARC. The presence of this protein in various soft tissues was detected by immunohistochemical technics. These results suggest a possibility that non-collagenous components in dentin may play regulatory roles in development, differentiation and calcification process of the dentin formation.