ROLE OF MATRIX VESICLES IN CALCIFICATION

基质囊泡在钙化中的作用

• 批准号: 3155014
• 负责人: ROY E WUTHIER
• 金额: $ 24.77万
• 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 1994-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3155014
• 关键词: Raman spectrometry; X ray crystallography; bone metabolism; calcification; calcium; calcium binding protein; calcium channel; calcium disorder; calcium metabolism; calcium phosphate; cartilage metabolism; chickens; chondrocytes; collagen; complementary DNA; connective tissue metabolism; electrolytes; epiphysis; extracellular matrix; gas chromatography; gel electrophoresis; infrared spectrometry; laboratory rabbit; lipid biosynthesis; membrane structure; messenger RNA; molecular cloning; monoclonal antibody; normal ossification; northern blottings; nuclear magnetic resonance spectroscopy; phosphorus; protein biosynthesis; radionuclides; radiotracer; tissue /cell culture; transport proteins; ultraviolet spectrometry; western blottings; Raman spectrometry; X ray crystallography; bone metabolism; calcification; calcium; calcium binding protein; calcium channel; calcium disorder; calcium metabolism; calcium phosphate; cartilage metabolism; chickens; chondrocytes; collagen; complementary DNA; connective tissue metabolism; electrolytes; epiphysis; extracellular matrix; gas chromatography; gel electrophoresis; infrared spectrometry; laboratory rabbit; lipid biosynthesis; membrane structure; messenger RNA; molecular cloning; monoclonal antibody; normal ossification; northern blottings; nuclear magnetic resonance spectroscopy; phosphorus; protein biosynthesis; radionuclides; radiotracer; tissue /cell culture; transport proteins; ultraviolet spectrometry; western blottings

Calcification is a biological process absolutely essential to the survival of all vertebrate organisms, including man. However, the unwanted deposition of mineral in vascular walls and valves contributes significantly to pathogenesis in arteriosclerosis. Despite its critical importance, the mechanism of calcification is not well understood. The long-term objective of the proposed research is to elucidate the mechanism of endochondral calcification. This calcifying system is not only critical to bone development, but also appears to be closely analogous to ectopic mineral deposition. The proposed research will explore the role of matrix vesicles (MV) structures now widely accepted as initiators of both normal calcification in cartilage and early bone formation, in the mechanism of mineral deposition. Attention will be focused on 1) the metabolism of Ca and Pi, and the characterization of very early mineral forms in MV during the induction of mineral formation, 2) characterization of key MV proteins involved in this process, 3) exploration of the relationship between MV and collagen in mineral deposition, and 4) utilization of specific inhibitors to elucidate the sequence of events in MV mineral deposition. Special attention will be directed towards: a) characterization of constitutive MV proteins [the newly-discovered lipid-dependent Ca2+-binding proteins, the water-soluble peripheral proteins, and the collagen-binding proteins] and their relationship to the induction of mineral formation by MV, b) characterization of mineral precursors formed during early stages of MV mineralization, and c) elucidating the effect of the electrolyte environment on MV mineralization. Epiphyseal growth plate cartilage from rapidly growing chickens will be used to provide an abundant source of actively calcifying material for isolation of cells and MV. Experimental methods will include: Tissue fractionation, cell culture, assays of MV 45Ca- and 32Pi-metabolism, protein purification (extraction, chromatography, electrophoresis) and characterization using amino acid analysis, peptide mapping and sequencing, immunology (polyclonal and monoclonal antibodies, Western blots) and molecular biology (mRNA isolation, Northern blots, cDNA library, cDNA cloning and sequencing). Spectroscopy (FTIR, NMR, UV), electron microscopy and x-ray diffraction will be used to characterize MV mineral phases.

钙化是生存绝对必要的生物学过程 在包括人在内的所有脊椎动物生物中。 但是，不需要的 矿物在血管壁和瓣膜中的沉积有贡献 在动脉粥样硬化中的发病机理显着。 尽管它很关键 重要的是，钙化的机制尚不清楚。 这 拟议的研究的长期目标是阐明机制 内侧软骨钙化。 这个钙化系统不仅关键 骨发育，但似乎也与异位相似 矿物沉积。 拟议的研究将探讨矩阵的作用 囊泡（MV）结构现在被广泛接受为两种正常的启动者 软骨和早期骨形成的钙化 矿物沉积。 注意将集中于1）CA的代谢 和PI，以及MV中非常早期矿物形式的表征 矿物形成的诱导，2）键MV蛋白的表征 参与此过程，3）探索MV与 矿物质沉积中的胶原蛋白，4）利用特定抑制剂 阐明MV矿物质沉积中的事件序列。 特别的 注意将指向：a）本构MV的表征 蛋白质[新发现的脂质依赖性Ca2+结合蛋白， 水溶性外周蛋白，以及胶原结合蛋白]和 它们与MV诱导矿物形成的关系，b） MV早期阶段形成的矿物前体的表征 矿化，c）阐明电解质的作用 MV矿化环境。 外周期生长板软骨 快速生长的鸡将用于提供丰富的来源 积极钙化材料，用于分离细胞和MV。 实验 方法将包括：组织分馏，细胞培养，MV的测定 45CA-和32PI​​代谢，蛋白质纯化（提取，，， 色谱，电泳）和使用氨基酸进行表征 分析，肽映射和测序，免疫学（多克隆和 单克隆抗体，蛋白质印迹）和分子生物学（mRNA 隔离，北印迹，cDNA文库，cDNA克隆和测序）。 光谱（FTIR，NMR，UV），电子显微镜和X射线衍射 将用于表征MV矿物相。