ROLE OF MATRIX VESICLES IN CALCIFICATION

细胞外基质囊泡被认为在 软骨、骨和牙本质中钙化的初始阶段，但它们的 其作用机制尚不清楚。该计划的目标是 建议的研究是为了阐明软骨内的机制。 钙化，特别强调基质小泡的作用。 我们将把注意力集中在1)决定因素上 细胞形成和释放基质小泡，2)续 它们的酶、蛋白质、矿物质形态和膜的表征 3)猪的序列和化学计量学的进一步研究 基质囊泡在矿物质诱导中的钙代谢 队形。特别是，研究的目的将是a)回避这一角色 磷酸钙诱导的膜融合，以及酸性 磷脂-钙-PI复合体，形成基质小泡，b) 后续基质中预装矿物需要量的确定 囊泡矿化，以及c)确定碱的作用 磷酸酶等囊泡蛋白的运输和积累 在基质囊泡介导的矿化过程中的磷和钙的变化。我们会 继续使用从长骨中长出的快速生长的骨骺软骨 鸡作为我们的实验组织。这一来源提供了丰富的， 用于分离细胞和基质小泡的活性钙化材料， 并研究它们的新陈代谢。本研究项目中使用的技术 将进行组织培养，细胞分离，45Ca-和32PI-代谢， 化学和物理方法：层析(柱层析、薄层、高效液相色谱、 2维等)、电泳法(PAGE、SDS-PAGE、等电点 聚焦)、酶消化(纯化、动力学分析)、光谱学(UV、 红外、核磁共振、MAS-核磁共振)、电子显微镜(透射式、扫描式、 冷冻断口)、X射线衍射仪等。

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.