Spectroscopy of Protein Mediation of Bone Mineralization

描述（由申请人提供）：该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由非胶原蛋白介导的三步序列（从无序磷酸钙到八磷酸钙（OCP）样矿物到碳酸磷灰石），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），并且这个过程可以实时跟踪。我们进一步假设，首次沉积的矿物通过ocp样中间体发育成碳酸磷灰石，并且这种矿物的转化是通过一层接一层的机制发生的，转化时间约为3小时。我们的测试系统将是胎鼠颅骨组织切片。对于这四种蛋白中的每一种，在培养过程中，以腺病毒为基础的蛋白在头颅组织切片上的过表达都会扰乱矿化。为了可视化感染的成骨细胞，我们将使用腺病毒编码所选的非胶原蛋白和绿色荧光蛋白（GFP）。GFP将作为受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的成骨细胞的位置。孵育后组织切片通过组织化学可视化来确认过表达蛋白的位置。相关拉曼/GFP荧光成像将确定新矿物与OPN的共定位。点突变将用于评估过表达蛋白中特定结构域缺失的影响。这些实验将由合成OCP和碳化磷灰石的模型化合物研究补充，包括粉末衍射模式的测量和密度函数理论的振动光谱带位置的计算。总的来说，该项目将使我们能够建立骨矿物从最初沉积的无序磷酸钙到最终稳定的磷灰石碳酸盐转变的机制和动力学。由此获得的信息对于指导开发治疗骨质疏松症和其他骨骼疾病的新合成代谢药物将是非常宝贵的。我们在这个资助周期中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矩阵和交联形成过程。公共卫生相关性：该建议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（包括骨质疏松症和骨遗传缺陷）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.