MINERALIZATION STUDIES RELATED TO ORAL BIOLOGY

拟议的研究将集中在基质囊泡（MV），这将发挥作用， 在牙齿和骨骼矿化中的作用。 MV是亚微观的， 细胞外，膜投资颗粒，启动钙化的 牙本质、生长软骨和发育中的骨骼。 我们的实验室参与了 MV的首次鉴定、分离和表征。 我们有 提供证据表明MV磷酸酶，包括碱性磷酸酶 (ALP)参与MV矿化。 此外，这些磷酸酶 是MV膜的组成部分，在MV膜中矿物质首次出现（在其内部 表面）表明ALP和其他成分的重要作用， MV膜启动钙化。 我们的具体目标是 为了增加对分子和结构的理解 组织的MV膜和SAP，重点是确定主要 组成性蛋白质，进一步测试碱性磷酸酶的功能， 孤立微血管钙化机制，探讨微血管钙化的调控机制 通过培养的软骨细胞和骨细胞在体外进行生物合成。 建议的新研究包括：1）研究ALP与MV的关系 膜使用ALP释放剂与EM细胞化学剂偶联， ALP免疫细胞化学定位; 2）“western”印迹分析， 鉴定MV的主要组成蛋白，并将这些蛋白指定为 MV膜或SAP; 3）研究离体的 大鼠MV检查ALP在支持钙化中的作用， 受ALP抑制剂或ALP释放剂或ALP增强剂干扰 三碘甲腺原氨酸、1，25-OH 2-D3和NaF等药物;以及4）MV的研究 通过MC 3 T3-E1大鼠成骨细胞在培养物中的生物发生。 的比较 由MC 3 T3-E1细胞产生的MV与来自原代软骨细胞培养物的MV 关于MV的产量、特征性主要蛋白质的存在以及 存款45 Ca的能力;和5）测试 可能的调节因素，如细胞周期或离子载体的阶段， 诱导Ca ~（2+）进入MV产生细胞。 这是一项关于牙齿和骨骼 开始形成矿化。 基质囊泡的新认识 钙化可以应用于广泛的主题，包括特定的 发生异常钙化的疾病。

The proposed study will focus on matrix vesicles (MVs) which will play a role in the mineralization of teeth and bones. MVs are sub-microscopic, extracellular, membrane-invested particles that initiate calcification of dentin, growth cartilage and developing bone. Our lab was involved in the first identification, isolation and characterization of MVs. We have provided evidence that MV phosphatases, including alkaline phosphatase (ALP), are involved in MV mineralization. Furthermore, these phosphatases are integral to the MV membrane where mineral first appears (at its inner surface) suggesting an important 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 major constituitive proteins, further testing of the function of ALP in the calcification mechanism of isolated MVs, and studying the regulation of MV biogenesis in vitro by cultured chondrocytes and bone cells. Proposed new studies include: 1) studying the relationship of ALP to the MV membrane using ALP releasing agents coupled with EM cytochemical and immunocytochemical localization of ALP; 2) A "western" blot analysis to identify major constitutive proteins of MVs and to assign these proteins to either MV membrane or sap; 3) studying in vitro calcification of isolated rat MVs to examine the role of ALP in supporting calcification, as perturbed by inhibitors of ALP or ALP releasing agents or by ALP-enhancing agents such as triiodothyronine, 1,25-OH2-D3 and NaF; and 4) studies of MV bio- genesis in culture by MC3T3-El rat osteoblast cells. A comparison of MVs produced by MC3T3-E1 cells versus MVs from primary chondrocyte cultures as to yield of MVs, the presence of characteristic major proteins, and the ability to deposit 45Ca; and 5) testing the effect on MV biogenesis of possible regulatory factors such as stage of the cell cycle or ionophore- induced Ca2+ entry into MV producing cells. This is a fundamental study of the mechanism by which dental and skeletal forms of mineralization are initiated. New knowledge of matrix vesicle calcification can be applied to a broad range of topics including specific diseases in which abnormal calcification occurs.