MINERALIZATION STUDIES RELATED TO ORAL BIOLOGY

拟议的研究将侧重于基质囊泡(MVS)，它将发挥一种 在牙齿和骨骼的矿化过程中起着重要作用。MV是亚微观的， 细胞外，膜包裹的颗粒，启动钙化 牙本质，生长软骨，发育骨。我们的实验室参与了 首次鉴定、分离和鉴定MVS。我们有 提供证据表明MV磷酸酶，包括碱性磷酸酶 (ALP)，参与MV成矿作用。此外，这些磷酸酶 与矿物质首次出现的MV膜(在其内部)是不可分割的 表面)表明了碱性磷酸酶和其他成分的重要作用 MV膜启动钙化。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，重点是识别主要 构成蛋白，进一步测试碱性磷酸酶在 游离血管钙化机制及血管钙化调控的研究 培养的软骨细胞和骨细胞的体外生物生成。 建议的新研究包括：1)研究ALP与MV的关系 用碱性磷酸酶释放剂结合EM细胞化学和 碱性磷酸酶的免疫细胞化学定位；2)免疫印迹分析 鉴定MVS的主要构成蛋白，并将这些蛋白分配给 MV膜或汁液；3)体外钙化研究 大鼠MVS检测碱性磷酸酶在支持钙化中的作用，AS 被碱性磷酸酶或碱性磷酸酶释放剂的抑制剂或碱性磷酸酶增强剂所干扰 三碘甲腺原氨酸、1，25-OH2-D3和NaF等药物；4)MV的研究 MC3T3-EL大鼠成骨细胞的体外生物发生。一种比较 MC3T3-E1细胞产生的微血管与软骨细胞原代培养产生的微血管的比较 关于MVS的产量、特征主要蛋白质的存在以及MVS 沉积45Ca的能力；5)测定其对MV生物发生的影响。 可能的调控因素，如细胞周期的阶段或离子载体- 诱导钙离子进入MV产生细胞。 这是对牙齿和骨骼 矿化形式开始形成。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 出现异常钙化的疾病。

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.