ROLE OF MATRIX VESICLES IN CALCIFICATION

钙化是生物生存必不可少的一种生物过程。 包括人类在内的所有脊椎动物。然而，不受欢迎的 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 重要的是，钙化的机制还不是很清楚。这个 拟议研究的长期目标是阐明这一机制。 软骨内钙化。这个钙化系统不仅对 对骨骼的发育，但也似乎与异位密切相似 矿物沉积。拟议的研究将探索矩阵的作用 囊泡(MV)结构现在被广泛认为是两种正常的 在软骨钙化和早期骨形成的机制中 矿物沉积。将重点关注1)钙的代谢 和pI，以及MV中非常早期矿物形态的特征 矿物形成的诱导，2)关键MV蛋白的特性 参与这一过程，3)探讨了MV与 矿物沉积中的胶原蛋白，以及4)特定抑制剂的利用 阐明MV矿物沉积过程中的事件顺序。特价 将重点关注：a)构成MV的特征 蛋白质[新发现的脂类依赖的钙结合蛋白， 水溶性外周蛋白和胶原结合蛋白]和 它们与MV，b)诱导矿物形成的关系 MV早期形成的矿物前体的特征 矿化，以及c)阐明电解液的影响 环境对MV成矿作用的影响。骨骺生长板软骨来源 快速生长的鸡将被用来提供丰富的 用于分离细胞和MV的活性钙化材料。实验 方法包括：组织分离、细胞培养、病毒检测。 ~(45)Ca和~(32)PI代谢，蛋白质纯化(提取， 色谱学、电泳学)和氨基酸表征 分析、肽图谱和测序、免疫学(多克隆和 单抗，免疫印迹)和分子生物学(信使核糖核酸 分离、Northern blots、cDNA库、cDNA克隆和测序)。 光谱学(FTIR、核磁共振、UV)、电子显微镜和X射线衍射 将被用来表征MV矿物相。

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.