ROLE OF MATRIX VESICLES IN CALCIFICATION

基质囊泡在钙化中的作用

• 批准号: 3155010
• 负责人: ROY E WUTHIER
• 金额: $ 17.65万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-01-01 至 1986-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3155010
• 关键词: Raman spectrometry; X ray crystallography; adenosinetriphosphatase; alkaline phosphatase; bone metabolism; calcification; calcium metabolism; calcium phosphate; cartilage; cartilage metabolism; chondrocytes; connective tissue metabolism; electron microscopy; epiphysis; extracellular matrix; freeze etching; gas chromatography; gel electrophoresis; infrared spectrometry; lipid biosynthesis; magnesium; membrane structure; normal ossification; nuclear magnetic resonance spectroscopy; paper chromatography; phospholipids; pyrophosphates; radiotracer; thin layer chromatography; ultraviolet spectrometry

Extracellular matrix vesicles are believed to play a key role in the initial stages of calcification in cartilage, bone and dentin, but their mechanism of action is still not well understood. The objective of the proposed research is to elucidate the mechanism of endochondral calcification with special emphasis on the role of matrix vesicles. Attention will be focused on 1) determining factors involved in the formation and release of matrix vesicles by cells, 2) continued characterization of their enzymes, proteins, mineral forms and membrane constituents, and 3) further study of the sequences and stoichiometry of Pi and Ca metabolism by matrix vesicles in the induction of mineral formation. In particular, studies will be aimed at a) eludicating the role of calcium phosphate-induced membrane fusion, and of acidic phospholipid-Ca-Pi complexes, in the formation of matrix vesicles, b) ascertaining the requirement of preloaded mineral in subsequent matrix vesicle mineralization, and c) determining the role of alkaline phosphatase, and other vesicle proteins, in the transport and accumulation of Pi and Ca during matrix vesicle-mediated mineralization. We will continue to use epiphyseal cartilage from long bones of rapidly growing chickens as our experimental tissue. This source provides abundant, actively calcifying material for isolation of cells and matrix vesicles, and studying their metabolism. Techniques used in this research project will be tissue culture, cell fractionation, 45Ca- and 32Pi-metabolism, chemical and physical methods: chromatography (column, thin-layer, HPLC, 2-dimensional, etc.), electrophoresis (PAGE, SDS-PAGE, isoelectric focusing), enzymolegy (purification, kinetic analyses), spectroscopy (UV, IR, NMR, MAS-NMR), electron microscopy (transmission, scanning, freeze-fracture), x-ray diffraction, etc.

细胞外基质囊泡被认为在 软骨、骨和牙本质钙化的初始阶段，但它们 作用机制仍不十分清楚。 该计划的目标 拟议的研究旨在阐明软骨内的机制 钙化特别强调基质囊泡的作用。 注意力将集中在 1) 涉及的决定因素 细胞基质囊泡的形成和释放，2) 继续 其酶、蛋白质、矿物质形式和膜的表征 成分，以及 3) 进一步研究 Pi 的序列和化学计量 和钙代谢通过基质囊泡诱导矿物质 形成。 特别是，研究的目的是：a) 阐明 磷酸钙诱导的膜融合和酸性 磷脂-Ca-Pi 复合物，在基质囊泡的形成中，b) 确定后续基质中预装矿物的要求 囊泡矿化，c) 确定碱性的作用 磷酸酶和其他囊泡蛋白的运输和积累 基质囊泡介导的矿化过程中 Pi 和 Ca 的含量。 我们将 继续使用来自快速生长的长骨的骨骺软骨 鸡作为我们的实验组织。 这个来源提供了丰富的、 用于分离细胞和基质囊泡的主动钙化材料， 并研究它们的新陈代谢。 本研究项目中使用的技术 将是组织培养、细胞分级、45Ca-和32Pi-代谢， 化学和物理方法：色谱法（柱、薄层、HPLC、 二维等）、电泳（PAGE、SDS-PAGE、等电 聚焦）、酶学（纯化、动力学分析）、光谱学（UV、 IR、NMR、MAS-NMR）、电子显微镜（透射、扫描、 冷冻断裂）、X射线衍射等。