Functions of Non-Collagenous Proteins in Dentin

已知牙本质基质中存在各种非胶原成分，例如磷酸蛋白、蛋白聚糖、骨连接素和血清α-2-HS-糖蛋白。这些成分可能与牙本质的钙化机制有关。本研究旨在分离、纯化和鉴定牙本质中的非胶原蛋白，以阐明其生理功能。并且，获得了以下结果。1)加入牙本质磷蛋白后，琼脂凝胶体系中磷酸钙沉淀量减少，但有明显的条带效应。去磷酸化的磷蛋白没有影响。该蛋白在乳牙和恒牙以及牙冠和牙根牙本质中的分布不同。牙本质形成过程中，牙本质中不含磷素。用成牙本质细胞孵育法和免疫印迹法鉴定牛磷酸化蛋白的生物合成。2)牙本质中的蛋白多糖进行了分析，比较在骨和软组织。阐明了牙本质及相关组织中低分子量蛋白多糖核心蛋白的结构和免疫学特性。在软组织中也发现了这种类型的蛋白聚糖。3)从成牙本质细胞mRNA制备cDNA，用抗骨连接素单克隆抗体克隆，并从cDNA的碱基序列中鉴定了牙本质骨连接素的氨基酸序列。该序列与1999年的序列相同。免疫组化法检测该蛋白在各种软组织中的表达。这些结果提示，牙本质中的非胶原成分可能在牙本质的发育、分化和钙化过程中起调节作用。

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.