SPECTROSCOPY OF PROTEIN MEDIATION OF BONE MINERALIZATION

描述(申请人提供)：这个项目的总体目标是通过使用实时相关的拉曼显微光谱/荧光成像方法来全面了解骨组织矿化的化学。我们的假设是，矿化遵循三个步骤(无序的磷酸钙到类八钙(OCP)矿物再到碳化的磷灰石)，这个过程是由非胶原蛋白介导的，包括骨唾液蛋白(BSP)、牙本质基质蛋白1(DMP1)、骨钙素(OC)和骨桥蛋白(OPN)，这个过程可以实时跟踪。我们进一步假设，首先沉积的矿物通过类OCP中间体发展成碳化磷灰石，并且这种矿物的转变是通过大约3小时的转化时间通过逐层机制发生的。我们的测试系统将是胎鼠颅骨组织切片。对于这四种蛋白质中的每一种，矿化都会被基于腺病毒的蛋白质在培养下的颅骨组织切片上的过度表达所干扰。为了可视化被感染的成骨细胞，我们将使用腺病毒来编码所选的非胶原蛋白和绿色荧光蛋白(GFP)。GFP将作为感染成骨细胞的荧光标记物。组织切片将通过近红外拉曼显微光谱连续监测12-24小时，使用荧光标记物识别过表达蛋白质的成骨细胞的位置。孵育后，组织切片进行组织化学观察，以确定过表达蛋白的定位。相关的拉曼/GFP荧光成像将建立新矿物和OPN的共定位。点突变将被用来评估在过度表达的蛋白质中删除特定结构域的效果。对合成OCP和碳化磷灰石的模型化合物研究将补充这些实验，包括测量粉末衍射图和用密度泛函理论计算振动光谱带位置。总体而言，该项目将使我们能够建立从最初沉积的无序磷酸钙到最终稳定的磷灰石碳酸盐的骨矿转化的机制和动力学。由此获得的信息将对指导治疗骨质疏松症和其他骨骼疾病的新合成代谢药物的开发具有无价的指导作用。我们在这一赠款周期中的成功将导致对更复杂的基质和交联链形成过程的实时测量的密切相关方法的扩展。与公共健康相关：该提案使用光学测量来跟踪骨矿物质在几个阶段的发展。众所周知，这一过程在代谢性骨病中起作用，包括骨质疏松症和骨的遗传缺陷。从这个项目中获得的信息将有助于为其中一些疾病设计新的治疗剂。

DESCRIPTION (provided by applicant): The overall aim of this project is a complete understanding of the chemistry of mineralization of bone tissue through use of real-time correlative Raman microspectroscopy/fluorescence imaging methods. Our hypothesis is that mineralization follows a three-step sequence (disordered calcium phosphate to octacalcium phosphate (OCP)-like mineral to carbonated apatite) that is mediated by non-collagenous proteins, including bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), osteocalcin (OC) and osteopontin (OPN) and that this process can be followed in real time. We further hypothesize that the development of first-deposited mineral into carbonated apatite proceeds through the OCP-like intermediate and that the transformation of this mineral occurs through a layer-by-layer mechanism over a conversion time of approximately 3 hours. Our test system will be fetal murine calvarial tissue sections. For each of the four proteins, mineralization will be perturbed by adenovirus-based overexpression the of protein to calvarial tissue sections under culture. To visualize the infected osteoblasts we will use adenoviruses that code for both the selected non-collagenous protein and green fluorescent protein (GFP). The GFP will serve as a fluorescent marker for infected osteoblasts. The tissue sections will be continuously monitored for 12-24 hours by near-infrared Raman microspectroscopy, using fluorescent markers to identify the locations of osteoblasts with overexpressed protein. After the incubation the tissue section will be visualized by histochemistry to confirm the loecation of overexpressed protein. Correlative Raman/GFP fluorescence imaging will establish co-localization of new mineral and OPN. Point mutations will be used to evaluate effects of the deletion of specific domains in the overexpressed proteins. These experiments will be complemented by model compound studies on synthetic OCP and carbonated apatites, including measurement of powder diffraction patterns and computation of vibrational spectroscopic band positions by density function theory. Overall, the project will allow us to establish the mechanism and kinetics of the bone mineral transformation from first deposited disorded calcium phosphate to final stable apatitic carbonate. The information so gained will be invaluable in guiding the development of new anabolic agents for treatment of osteoporosis and other skeletal diseases. Our success in this grant cycle will lead to an extension to closely related methods for real-time measurement of the more complicated processes of matrix and cross-link formation. PUBLIC HEALTH RELEVANCE: The proposal uses optical measurements to follow development of bone mineral through several stages. It is known that this process malfunctions in metabolic bone diseases, including osteoporosis and in genetic defects of bone. The information gained in this project will help in the design of new therapeutic agents for some of these diseases.