MINERALIZATION STUDIES RELATED TO ORAL BIOLOGY

拟议的研究将侧重于发挥作用的基质囊泡(MVS) 在牙齿矿化过程中起着重要作用。基质小泡是 亚显微、胞外、膜包被颗粒 作为牙本质矿化的初始部位，以及 其他口腔组织，包括软骨和骨。我们的实验室是 参与最初的MVS识别和他们的 随后的分离和部分表征。我们有 提供证据表明基质囊泡磷酸酶(包括 ATPase、焦磷酸酶和碱性磷酸酶)参与 矿化作用。此外，这些磷酸酶是相关的 与囊泡膜结合，保存后可溶解 酶的活性，并重组成囊泡 钙沉积活性的恢复。在工作中得到支持 目前我们所拥有的资助：1)开发了一种体外方法来 在允许的条件下研究分离的MV钙化 羟基磷灰石(HA)矿物沉积；2)研制了一种 抗MV碱性磷酸酶(ALPase)抗体，并用其 纯化碱性磷酸酶至化学均一的量永不 以前可用的；3)在第一次使用单抗 尝试在细胞膜的EM水平免疫定位ALPase 细胞和MVS；以及4)设计了一种哺乳动物细胞培养系统 遵循MV的生物发生和释放(可能是从质膜) MV体外钙化。 拟议的新研究包括：1)试图确定 ALPase在MV膜中的定位和定位 使用新抗体的免疫细胞化学，以及通过 特定的酶、萃取剂和洗涤剂的应用 表明MV ALPase是否是跨膜蛋白；2)和 重组MV-ALPase为蛋白脂质体的尝试 恢复酶活性和钙化能力；3)使用 哺乳动物软骨细胞培养检测MV生物发生的研究 更多细节，包括可能的新陈代谢控制。 这是一项关于牙本质和 矿化的骨架形式就产生了。新知识 基质囊泡钙化可广泛应用于 主题包括特定疾病状态，哪些异常 就会发生钙化。

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.