ROLE OF MATRIX VESICLES IN CALCIFICATION

基质囊泡在钙化中的作用

基本信息

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
项目类别：
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

项目摘要

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.

钙化是一个对于生存绝对必要的生物过程所有脊椎动物，包括人类。然而，不想要的矿物质在血管壁和瓣膜中的沉积有助于对动脉硬化的发病机制有重要意义。尽管它很关键重要性，钙化的机制尚不清楚。这拟议研究的长期目标是阐明其机制软骨内钙化。这个钙化系统不仅至关重要与骨骼发育有关，但也与异位密切相似矿物沉积。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正常和软骨钙化和早期骨形成的机制矿物沉积。注意力将集中在1）Ca的代谢上和 Pi，以及 MV 期间非常早期矿物形态的表征矿物质形成的诱导，2) 关键 MV 蛋白的表征参与这个过程，3）探索MV与矿物质沉积中的胶原蛋白，以及 4) 特定抑制剂的利用阐明 MV 矿物沉积的事件顺序。特别的注意力将集中在： a) 本构 MV 的表征蛋白质 [新发现的脂质依赖性 Ca2+ 结合蛋白，水溶性外周蛋白和胶原蛋白结合蛋白]和它们与 MV 诱导矿物质形成的关系，b) MV 早期形成的矿物前体的表征矿化，c) 阐明电解质的影响 MV 成矿环境。骨骺生长板软骨来自快速生长的鸡将被用来提供丰富的资源用于分离细胞和 MV 的主动钙化材料。实验性的方法包括：组织分离、细胞培养、MV 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、肽图谱和测序、免疫学（多克隆和单克隆抗体、蛋白质印迹）和分子生物学（mRNA 分离、Northern 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。