ROLE OF MATRIX VESICLES IN CALCIFICATION

细胞外基质囊泡被认为在 在软骨、骨和牙本质中开始钙化，但其 其作用机制尚不完全清楚。建议的目标是 研究旨在阐明软骨内钙化的机制 特别强调基质小泡的作用。注意力将集中在 关于1)基质形成和释放的决定因素 细胞的囊泡，2)继续表征它们的酶， 蛋白质、矿物质形态和膜成分；3)进一步研究 基质囊泡对钙和磷的代谢作用 矿物形成。研究的目的将是a)阐明 局部营养因素对能量代谢的影响及其对细胞的影响 钙代谢，b)确定细胞钙代谢对 可钙化基质囊泡的形成，c)进一步阐明其作用 碱性磷酸酶及其相关蛋白在矿物质诱导中的作用 由基质小泡形成。我们将继续使用骨盆生长 以快速生长鸡长骨板软骨为来源 我们的组织和细胞。这一来源提供了丰富的、积极的 用于分离细胞和基质小泡的钙化材料 研究他们的新陈代谢。使用的技术将是组织培养、细胞 分级、~(45)Ca和~(32)PI代谢、化学和物理方法： 层析(柱层、薄层、高效液相、二维等) 电泳法(PAGE、SDS-PAGE、等电聚焦)、酶学 (提纯、动力学分析)、光谱(UV、IR、核磁共振、MAS-核磁共振)、 电子显微镜(透射式、扫描式、冷冻断裂式)、x射线 绕射等。

Extracellular matrix vesicles are believed to play a key role in the initiation of calcification in cartilage, bone and dentin, but their mechanism of action is not fully 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 metabolism of Ca and Pi by matrix vesicles during the induction of mineral formation. Studies will be aimed at a) elucidating the role of local nutritional factors on energy metabolism and its effect on cellular Ca metabolism, b) determining the effect of cellular Ca metabolism on the formation of calcifiable matrix vesicles, c) further elucidating the role of alkaline phosphatase and associated proteins in the induction of mineral formation by matrix vesicles. We will continue to use epiphyseal growth plate cartilage from long bones of rapidly growing chickens as the source of our tissue and cells. This source provides abundant, actively calcifying material for isolation of cells and matrix vesicles, and for studying their metabolism. Techniques used 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), enzymology (purification, kinetic analyses), spectroscopy (UV, IR, NMR, MAS-NMR), electron microscopy (transmission, scanning, freeze-fracture), x-ray diffraction, etc.