MATRIX VESICLES AND CALCIFICATION

基质囊泡和钙化

• 批准号: 6164017
• 负责人: ROY E WUTHIER
• 金额: $ 27.29万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-01-01 至 2002-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6164017
• 关键词: annexins; bioengineering /biomedical engineering; biomaterial development /preparation; biomimetics; calcification; calcium ion; chickens; chondrocytes; computer simulation; extracellular matrix proteins; laboratory rabbit; lipid metabolism; liposomes; magnesium ion; membrane transport proteins; molecular cloning; molecular dynamics; normal ossification; nuclear magnetic resonance spectroscopy; phospholipase A2; phospholipids; phosphorus; protein biosynthesis; protein reconstitution; protein sequence; protein structure function; sodium ion; zinc

The long-range goal of this research is to understand how endochondral calcification, a process vital for skeletal development and fracture healing, works. Because isolated matrix vesicles (MV) initiate calcification by acquiring large amounts of Ca2+ and Pi, they make an excellent model on which to base the fabrication of clinically useful biomaterials for stimulating bone healing. Major progress was made during the past grant period in characterizing the mechanism of MV mineralization. We have identified and characterized the Ca2+ transporter, made progress in purifying the MV phospholipase A2 and have laid important groundwork in cloning the Pi transporter. We have uncovered important information on MV lipid metabolism and in characterizing the initial mineral phase of MV, and have done substantial pilot work on reconstitution of the nucleational core and formation of synthetic MV. Building on these findings, our major goals for the next grant period are to elucidate how these lipids, proteins and electrolytes interact to trigger MV formation and mineralization. Since we have already characterized the Ca2+ porter, lipid, and electrolyte components of the nucleational complex, we will now characterize the MV Pi ion porter. Based on the finding of major changes in lipid composition during MV mineralization, we will now characterize two enzymes that appear to be responsible for these changes. During this past grant period we have also acquired a large body of spectroscopic data on the nature of the nucleational complex. We will now use this data to guide us in our reconstitution of the synthetic complex, as well as to deduce its structure using molecular simulations. Collectively, this body of information will allow us to precisely construct an implantable biomimetic material for stimulating bone healing. Our specific aims are: 1) to characterize three new MV proteins that appear to be critical to MV function, 2) to further elucidate MV formation, minerals, and metabolism, and 3) to reconstitute and simulate MV structure and function. Specifically we will characterize critical interactions that occur between the annexins, PS, Ca2+ and Pi during MV formation. Also the nature of the first mineral phase formed by MV and changes in MV lipid composition that accompany mineralization will be examined. The MV Na+-dependent Pi-transporter, the phospholipase A2 and the phospholipid base-exchange enzyme will be identified, cloned and sequenced, and their properties characterized. The MV nucleational complex will then be synthesized, characterized, and modeled by computer simulation. Finally, with this information in hand, functional MV will be reconstituted and tested for bone-healing properties.

这项研究的长期目标是了解软骨内如何 钙化是骨骼发育和骨折的关键过程 治愈，很管用。因为孤立的基质小泡(MV)启动 钙化通过获得大量的钙和磷，它们形成了 优秀的模型，为临床有用的产品的制作奠定了基础 用于促进骨愈合的生物材料。取得了重大进展。 在过去的授权期内对MV的机制进行表征 矿化作用。我们已经鉴定并表征了钙离子 转运蛋白，在纯化MV磷脂酶A2方面取得了进展，并已 为克隆PI转运蛋白奠定了重要基础。我们有 发现了关于MV脂代谢和在 表征了MV的初始矿物相，并已经做了 关于重建核核心和核的实质性试点工作 合成MV的形成。在这些发现的基础上，我们的主要目标是 下一个授权期是为了阐明这些脂质、蛋白质 和电解质相互作用，触发MV的形成和成矿。 因为我们已经确定了钙离子转运体、脂质和 成核复合体的电解液成分，我们现在将 对MV PI离子转运体进行表征。根据少校的调查结果 MV成矿过程中脂质成分的变化，我们现在将 描述两种似乎对此负责的酶的特征 改变。在过去的赠款期间，我们还获得了大量的 关于核复合体性质的大量光谱数据。 我们现在将使用这些数据来指导我们重建 合成的络合物，以及用分子推导其结构 模拟。总而言之，这些信息将使我们能够 精确构建一种可植入的仿生材料来刺激 骨愈合。我们的具体目标是：1)表征三个新的MV 看似对MV功能至关重要的蛋白质，2)进一步 阐明MV的形成、矿物质和代谢，以及3)重建 并对MV的结构和功能进行了仿真。具体来说，我们将 描述发生在膜联蛋白、PS、 花叶病毒形成过程中的Ca~(2+)和PI。也是第一种矿物的性质 MV形成的时相及伴随的MV脂组成变化 矿化作用将被研究。MV依赖于Na+的PI转运体， 磷脂酶A2和磷脂碱基交换酶将 鉴定、克隆和测序，并对它们的性质进行了表征。 然后将合成、表征和表征MV核复合体 通过计算机模拟进行建模。最后，有了这个信息， 功能性MV将被重组并进行骨愈合测试 属性。