MINERALIZATION STUDIES RELATED TO ORAL BIOLOGY

拟议的研究将集中在基质囊泡(MVS)上，它在 在牙齿和骨骼的矿化过程中起到启动作用。MV是 亚微观、细胞外、膜包裹的颗粒 牙本质中钙化的起始部位、生长软骨和 发育中的骨骼。我们的实验室参与了第一次鉴定， MVS的分离和鉴定。我们和其他人提供了 证据表明MV磷酸酶，包括碱性磷酸酶(ALP)，是 参与MV成矿作用。此外，这些磷酸酶是 整合到MV膜中，矿物第一次出现在膜下 提示碱性磷酸酶和MV的其他组成部分起着关键作用 启动钙化的膜。我们的具体目标是直接的 朝着增加对分子和结构的理解 MV膜和汁液的组织，重点是识别和 定位主要构成蛋白，检测病毒功能 磷酸酶在分离MVS钙化机制中的作用及研究 培养的骨髓细胞和骨髓对MV生物发生的调控 骨祖细胞。 拟议的新研究包括：1)进一步分析 分离的MVS完成主要蛋白质的鉴定；2) 膜与囊泡中MV主要成分的归属 应用高分辨率EM免疫定位结合生化的SAP 表面活性物质对膜结合蛋白的释放 洗涤剂的总释放量；3)进一步表征 MV磷酸酶在全软骨MVS矿化中的作用 在骨细胞或骨髓成骨祖细胞培养中产生MVS。 已知可增加MV的药物，如1,250H2D3、地塞米松或氟化物 碱性磷酸酶的活性，将被检测为对MV钙化的积极作用； 4)进一步的研究将通过以下方式来表征MV的生物发生机制 培养的大鼠软骨细胞、成骨细胞和成骨细胞 经已知促进骨化的药物修饰的骨髓基质 糖皮质激素、氟化物和维甲酸的体外分化 酸。 这是对牙齿和骨骼 矿化形式开始形成。关于矩阵的新知识 囊泡钙化可以应用于广泛的主题，包括 出现异常钙化的特定疾病。

The proposed study will focus on matrix vesicles (MVs) which play an initiating role in the mineralization of teeth and bones. MVs are submicroscopic, extracellular, membrane-invested particles that serve as the initial site of calcification in dentin, growth cartilage and developing bone. Our lab was involved in the first identification, isolation and characterization of MVs. We and others have provided evidence that MV phosphatases, including alkaline phosphatase (ALP), are involved in MV mineralization. Furthermore, these phosphatases are integrated into the MV membrane under which mineral first appears suggesting a critical role for ALP and other components of the MV membrane in initiating calcification. Our specific aims are directed toward an increased understanding of the molecular and structural organization of the MV membrane and sap with emphasis on identifying and localizing major constitutive proteins, testing the function of MV phosphatases in the calcification mechanism of isolated MVs, and studying the regulation of MV biogenesis by cultured bone cells and marrow osteoprogenitor cells. Proposed new studies include: 1) further analysis of the composition of isolated MVs to complete the identification of major proteins; 2) assignment of major MV constituents to the membrane versus the vesicle sap using high resolution EM immunolocalization coupled with biochemical release of membrane-attached proteins by surface active agents versus total release by detergents; 3) further characterization of the role of MV phosphatases in the mineralization of MVs from whole cartilage versus MVs generated in cultures of bone cells or marrow osteoprogenitor cells. Agents such as 1,250H2D3, dexamethasone, or fluoride, known to boost MV ALP activity, will be tested for a positive effect on MV calcification; 4) further studies will characterize the mechanism of MV biogenesis by cultured rat chondrocytes, osteoblasts and osteoprogenitor cells of the marrow stroma as modified by agents known to promote osseous differentiation in vitro including glucocorticoids, fluoride and retinoic acid. This is a fundamental study of the mechanism by which dental and skeletal forms of mineralization are initiated. New knowledge of the matrix vesicle calcification can be applied to a broad range of topics including specific diseases in which abnormal calcification occurs.