MINERALIZATION STUDIES RELATED TO ORAL BIOLOGY

与口腔生物学相关的矿化研究

• 批准号: 3219334
• 负责人: HARRISON CLARKE ANDERSON
• 金额: $ 6.97万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-03-01 至 1989-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3219334
• 关键词: adenosinetriphosphatase; alkaline phosphatase; autoradiography; bone; calcification; cartilage; dentin; deoxycholate; electron microscopy; immunocytochemistry; pyrophosphates; radiotracer; tissue /cell culture; tooth

The proposed study will focus on matrix vesicles (MVs) which play a role in the mineralization of teeth. Matrix vesicles are submicroscopic, extracellular, membrane invested particles which serve as the initial loci of mineralization of dentin, and of other oral tissues including cartilage and bone. Our lab was involved in the original identification of MVs and in their subsequent isolation and partial characterization. We have provided evidence that matrix vesicle phosphatases (including ATPase, pyrophosphatase and alkaline phosphates) are involved in mineralization. Furthermore, these phosphatases are associated with the vesicle membrane and can be solubilized with preservation of enzymatic activity and reconstituted into vesicles with restitution of calcium-depositing activity. ln work supported by the present grant we have: 1) Developed an in vitro method to study isolated MV calcification under conditions which allow hydroxyapatIte (HA) mineral deposition; 2) Developed a monoclonal antibody against MV alkaline phosphatase (ALPase), and used it to purify ALPase to chemical homogeneity in quantities never previously available; 3) Used the monoclonal antibody in a first attempt to iummunolocalize ALPase at EM levels in membranes of cells and MVs; and 4) Devised a mammalian cell culture system to follow MV biogenesis and release (presumably from plasma membrane) and MV calcification in vitro. Proposed new studies include: 1) An attempt to determine the location and orientation of ALPase within the MV membrane by immunocytochemistry using new antibodies, and biochemically by application of specific enzymes, extractants and detergents which indicate whether MV ALPase is a transmembrane protein; 2) An attempt to reconstitute MV ALPase into proteoliposomes with restoration of enzyme activity and calcifiability; and 3) The use of mammalian chondrocyte cultures of examine MV biogenesis in greater detail, including possible metabolic controls. This is a fundamental study of the mechanism by which dentinal and skeletal forms of mineralization are brought about. New knowledge of matrix vesicle calcification can be applied to a broad range of topics including specific disease states which abnormal calcification occurs.

拟议的研究将重点介绍播放的基质囊泡（MV） 在牙齿矿化中的作用。 基质囊泡是 亚镜，细胞外的膜投资颗粒，这些颗粒 作为牙本质矿化的初始基因座， 其他口腔组织，包括软骨和骨骼。 我们的实验室是 参与了MVS的原始识别及其 随后的隔离和部分表征。 我们有 提供了基质囊泡磷酸酶的证据（包括 ATPase，焦磷酸酶和碱性磷酸盐）参与 矿化。 此外，这些磷酸酶是相关的 用囊泡膜，可以通过保存溶解 酶活性，并将其重构为囊泡 恢复钙沉积活性。 由支持 我们拥有的目前赠款：1）开发了一种体外方法 研究在条件下孤立的MV钙化允许 羟基磷灰石（HA）矿物沉积； 2）开发了单克隆 抗MV碱性磷酸酶（ALPase）的抗体，并将其用于 净化藻类的化学同质性量永远不会 以前可用； 3）在第一个中使用单克隆抗体 尝试在膜中以EM级别的ALPASE iummunolocalocalocalocalcase Inpase 细胞和MV； 4）将哺乳动物细胞培养系统设计为 遵循MV生物发生并释放（大概是质膜） 和MV钙化体外。 拟议的新研究包括：1）尝试确定 MV膜中ALPase的位置和方向 使用新抗体进行免疫细胞化学，并通过生化 应用特定酶，萃取剂和洗涤剂的应用 指示MV alpase是否是跨膜蛋白； 2） 试图用 恢复酶活性和钙化性； 3）用途 检查MV生物发生的哺乳动物软骨细胞培养物 更大的细节，包括可能的代谢控制。 这是对牙本质和牙本质机制的基本研究 提出了矿化的骨骼形式。 新知识 基质囊泡钙化可以应用于广泛的 主题包括异常的特定疾病状态 钙化发生。