MINERALIZATION STUDIES RELATED TO ORAL BIOLOGY

与口腔生物学相关的矿化研究

• 批准号: 3219336
• 负责人: HARRISON CLARKE ANDERSON
• 金额: $ 15.01万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-03-01 至 1992-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3219336
• 关键词: 1,25 dihydroxycholecalciferol; alkaline phosphatase; calcification; calcium; cartilage; cell growth regulation; chymotrypsin inhibitor; dentin; deoxycholate; electron microscopy; fluorine; gel electrophoresis; histochemistry /cytochemistry; immunocytochemistry; laboratory rat; membrane proteins; osteoblasts; phosphomonoesterases; proteins; radioassay; tissue /cell culture; tooth; triiodothyronine; western blottings

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.

拟议的研究将集中于基质囊泡（MV），该囊泡将发挥作用 在牙齿和骨骼的矿化中作用。 MV是亚显微镜， 启动钙化的细胞外膜投资颗粒 牙本质，生长软骨和发育骨骼。 我们的实验室参与了 MV的首次识别，隔离和表征。 我们有 提供的证据表明MV磷酸酶，包括碱性磷酸酶 （ALP）参与MV矿化。 此外，这些磷酸酶 是首次出现矿物的MV膜不可或缺的一部分（在其内部 表面）提出了ALP和其他组件的重要作用 MV膜启动钙化。 我们的具体目标是指导的 促进对分子和结构的了解 MV膜和SAP的组织，重点是确定专业 组成蛋白，进一步测试ALP在 分离的MV的钙化机制，并研究MV的调节 培养的软骨细胞和骨细胞在体外生物发生。 拟议的新研究包括：1）研究ALP与MV的关系 使用ALP释放剂与EM细胞化学和 ALP的免疫细胞化学定位； 2）“西部”印迹分析 识别MVS的主要组成蛋白，并将这些蛋白质分配给 MV膜或SAP； 3）研究分离的体外钙化 大鼠MV检查ALP在支持钙化中的作用，AS 受ALP或ALP释放剂的抑制剂或ALP增强的抑制剂扰动 诸如Triiodothyronine，1,25-OH2-D3和NAF之类的药物； 4）MV的研究 MC3T3-EL大鼠成骨细胞在培养中的生物发生。 比较 由MC3T3-E1细胞产生的MV与原发性软骨细胞培养物的MV 至于MV的产量，具有特征性主要蛋白质的存在和 能够存入45CA； 5）测试对MV生物发生的影响 可能的调节因素，例如细胞周期或离子载体 - 诱导Ca2+进入MV产生细胞。 这是对牙齿和骨骼的机制的基本研究 开始矿化形式。 基质囊泡的新知识 钙化可以应用于广泛的主题，包括特定的主题 发生异常钙化的疾病。