SPECTROSCOPY OF PROTEIN MEDIATION OF BONE MINERALIZATION

描述（由申请人提供）：本项目的总体目标是通过使用实时相关拉曼显微光谱/荧光成像方法，全面了解骨组织矿化的化学过程。我们的假设是，矿化遵循一个三步序列（无序磷酸钙磷酸八钙（OCP）样矿物质碳酸磷灰石），这是由非胶原蛋白，包括骨涎蛋白（BSP），牙本质基质蛋白1（DMP 1），骨钙素（OC）和骨桥蛋白（OPN）介导的，这个过程可以在真实的时间。我们进一步假设，第一沉积的矿物发展成碳酸磷灰石的过程中，通过OCP样中间体，这种矿物的转化发生通过一个层一层的机制，在约3小时的转换时间。我们的测试系统将是胎鼠颅骨组织切片。对于四种蛋白质中的每一种，矿化将被基于腺病毒的蛋白质向培养下的颅骨组织切片的过表达所干扰。为了观察感染的成骨细胞，我们将使用编码所选非胶原蛋白和绿色荧光蛋白（GFP）的腺病毒。GFP将作为感染成骨细胞的荧光标记。将通过近红外拉曼显微光谱法连续监测组织切片12-24小时，使用荧光标记物鉴定蛋白质过表达的成骨细胞的位置。孵育后，通过组织化学观察组织切片，以确认过表达蛋白的定位。相关的拉曼/GFP荧光成像将建立新矿物质和OPN的共定位。将使用点突变来评价在过表达蛋白质中缺失特定结构域的影响。这些实验将补充模型化合物的研究合成OCP和碳酸磷灰石，包括测量粉末衍射图案和计算振动光谱带的位置密度泛函理论。总的来说，该项目将使我们能够建立骨矿物质从最初沉积的无序磷酸钙到最终稳定的磷灰石碳酸盐转化的机制和动力学。所获得的信息将是非常宝贵的指导新的合成代谢药物治疗骨质疏松症和其他骨骼疾病的发展。我们在这一资助周期的成功将导致扩展到密切相关的方法，用于实时测量更复杂的基质和交联形成过程。公共卫生相关性：该提案使用光学测量方法通过几个阶段跟踪骨矿物质的发育。已知该过程在代谢性骨疾病（包括骨质疏松症）和骨的遗传缺陷中发生故障。在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药物。

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.