ROLE OF MATRIX VESICLES IN CALCIFICATION

细胞外基质囊泡被认为在细胞凋亡中起关键作用。 软骨、骨和牙本质钙化的初始阶段， 其作用机制尚不清楚。 的目的 建议的研究是阐明内软骨炎的机制， 钙化，特别强调基质囊泡的作用。 注意力将集中在1）参与的决定因素 细胞形成和释放基质囊泡，2）继续 它们的酶、蛋白质、矿物质形式和膜的表征 3）进一步研究了Pi的序列和化学计量 基质囊泡在诱导矿物质代谢中的作用 阵 特别是，研究的目的将是a）逃避的作用， 磷酸钙诱导的膜融合，以及 磷脂-Ca-Pi复合物，在基质囊泡的形成中，B） 确定后续基质中预载矿物的要求， 囊泡矿化，和c）确定碱性的作用 磷酸酶和其他囊泡蛋白，在运输和积累 基质囊泡介导的矿化过程中的Pi和Ca。 我们将 继续使用快速生长的长骨中的骺软骨 鸡作为实验组织 这个来源提供了丰富的， 用于分离细胞和基质囊泡的活性钙化材料， 研究它们的新陈代谢 本研究项目中使用的技术 将是组织培养，细胞分级，45 Ca和32 Pi代谢， 化学和物理方法：色谱法（柱，薄层，HPLC， 2维等），电泳（PAGE，SDS-PAGE，等电 聚焦），酶学（纯化，动力学分析），光谱学（UV， IR，NMR，MAS-NMR），电子显微镜（透射，扫描， 冷冻断裂）、X射线衍射等。

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.