PROTEOGLYCANS AND OSTEOPONTIN IN BONE FORMATION

这项建议的具体目的是：1)详细分析 骨蛋白多糖的化学和生物合成，2)用途 免疫定位实验中的抗体，以及3)评估 1，25-二羟基维生素D3的分子机制 似乎调节骨桥蛋白的生物合成。前两个目标 依赖经典的生化和免疫学方法，而 最后一个目标在某种程度上依赖于 分子生物学。申请者有使用这些工具的经验 技术，然而，这项研究事业的资金 发展奖将极大地促进他的研究事业 通过允许在这一领域进行更广泛的培训来促进发展。 这项研究所基于的主要假设是 矿化结缔组织的非胶原性成分为 与组织的形成密切相关。此外， 对其化学成分、定位和性质的认识 需要生物合成的调节机制才能 具体的、功能性的角色可以被阐明。在这份提案中，我们 我将讨论上述领域，因为它们与 蛋白多糖和骨桥蛋白。所采取的方法是将 这些成分的化学含量，验证它们的纯度和 用它们作为抗原。这些抗体将用于 免疫定位研究和免疫分析中的试剂。我们 将探索大鼠骨骼中蛋白多糖生物合成的动力学， 在ROS17/2.8细胞和大鼠颅骨成骨细胞培养中。 我们将评估1，25- 双羟基维生素D3对骨桥蛋白的调节作用 生物合成。

The specific aims of this proposal are 1) to analyze in detail the chemistry and biosynthesis of bone proteoglycans, 2) to use antibodies in immunolocalization experiments, and 3) to evaluate the molecular mechanism by which 1,25-dihydroxyvitamin D3 appears to regulate osteopontin biosynthesis. The first two aims depend on classical biochemical and immunological methods while the last aim relies, to some extent, on the techniques of molecular biology. The applicant has some experience using these techniques, however, the funding of this Research Career Development Award would greatly enhance his research career development by allowing more extensive training in this area. The major hypothesis upon which this research is based is that non-collagenous components of mineralized connective tissues are intimately involved in the formation of the tissues. Furthermore, an understanding of the chemial composition, localization and regulatory mechanisms of biosynthesis are required before specific, functional roles can be elucidated. In this proposal we will address the aforementioned areas as they relate to proteoglycans and osteopontin. The approach taken is to isolate chemical amounts of these components, verify their purity and use them as antigens. The antibodies will be used in immunolocalization studies and as reagents in immunoassays. We will explore the kinetics of proteoglycan biosynthesis in rat bone, in ROS 17/2.8 cells and in cultures of rat calvarial osteoblasts. We will evaluate the role and mechanism that 1,25- dihydroxyvitamin D3 may have in the regulation of osteopontin biosynthesis.