MINERALIZATION STUDIES RELATED TO ORAL BIOLOGY

拟议的研究将集中在基质囊泡（MV）， 在牙齿矿化中的作用。 基质囊泡是 亚显微镜下的细胞外膜包埋颗粒， 作为牙本质矿化的起始位点， 其他口腔组织包括软骨和骨。 我们的实验室 参与最初确定机动车辆及其 随后分离和部分表征。 我们有 提供的证据表明，基质囊泡磷酸酶（包括 ATP酶、焦磷酸酶和碱性磷酸酶）参与 成矿 此外，这些磷酸酶与 与囊泡膜，并可以溶解保存 并重组成囊泡， 钙沉积活性的恢复。 In work supported支持by 目前的赠款，我们有：1）开发了一种体外方法， 在允许的条件下研究孤立的MV钙化 羟基磷灰石（HA）矿物沉积; 2）开发了一种单克隆 抗MV碱性磷酸酶（ALP 13）的抗体，并将其用于 将氧化铝纯化至化学均质， 3）在第一次试验中使用单克隆抗体， 试图在EM水平免疫定位ALcasein， 细胞和MV;以及4）设计哺乳动物细胞培养系统， 遵循MV生物发生和释放（推测来自质膜） 和MV钙化。 建议的新研究包括：1）试图确定 在MV膜内的定位和方向， 免疫细胞化学使用新的抗体，生物化学， 应用特定的酶、提取剂和洗涤剂， 表明MV ALP 1是否是跨膜蛋白; 2） 尝试将MV ALGORIX重组为蛋白脂质体， 酶活性和可钙化性的恢复;和3）使用 的哺乳动物软骨细胞培养物中检测MV的生物发生， 更多细节，包括可能的代谢控制。 这是一项基础性的研究， 形成了矿化的骨架形式。 新知识 的基质囊泡钙化可以应用于广泛的 主题包括特定的疾病状态， 发生钙化。

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.