Osteocytes as Dynamic Cells

描述（由申请人提供）：骨细胞占骨细胞的90%以上。然而，由于难以在体内获得这些细胞或将其分离用于体外研究，因此对其功能知之甚少。虽然骨细胞通常被视为一种不活跃的细胞，但越来越多的证据表明，这些细胞对机械负荷有反应，并能够改变其局部环境，这表明它们可能比以前认为的更活跃。使用转基因小鼠，其中绿色荧光蛋白（GFP）的牙本质基质蛋白-1（DMP 1）启动子控制下的骨细胞为目标，我们进行了时间推移动态成像研究活骨细胞在颅骨器官培养。令人惊讶的是，这些研究表明，骨细胞远非静态细胞，而是高度动态的。骨细胞在骨陷窝内的胞体扩张和收缩，树突伸展和收缩。骨细胞之间的树突状连接和与骨表面上的运动细胞似乎是短暂的，与连接的建立和破坏。这些观察结果提出了树突，而不是永久的细胞间连接，可能是动态的结构，可以改变刺激影响骨细胞功能的可能性。提出的研究的中心假设是骨内的骨细胞是高度动态的细胞，它们的细胞体和树突运动对其功能和嵌入过程至关重要。为了验证这一假设，将使用多学科的方法，包括使用转基因小鼠模型的动态成像的活骨细胞和成骨细胞，连同细胞体和树突运动的计算分析。在目标1中，我们将使用最先进的成像技术来确定骨细胞及其树突在活体骨外植体中的动态特性。我们将确定这些变化是否随年龄和/或响应已知影响骨细胞功能的外部刺激，如缺氧和前列腺素E2。将确定细胞运动和肌动蛋白聚合/解聚抑制剂对作为骨细胞功能终点的间隙连接信号传导的影响。在目标2中，我们将使用骨形成器官培养模型动态成像成骨细胞嵌入和分化成骨细胞。这将使用分别由Col 1a 1和DMP 1启动子驱动的在成骨细胞中表达DsRed和在骨细胞中表达GFP的小鼠的颅骨来完成。还将对矿物质沉积进行成像，以确定成骨细胞嵌入/分化的动力学如何与矿化相结合。将确定细胞运动和肌动蛋白聚合/解聚抑制剂对包埋和矿化的影响。这些探索性研究的成功完成将开发新的和创新的模型，以促进骨细胞功能的研究，提供成骨细胞向骨细胞转化的过程的基本见解，并提供一个新的范式，将骨细胞树突视为动态交互结构。这将对我们理解骨细胞在正常骨和疾病（如骨质疏松症和骨软化症）中的作用产生重大影响。这些研究将在1-2年内为RO 1应用奠定基础。这项研究与公共卫生有关，因为它将为骨细胞的功能提供非常新颖的见解，骨细胞是骨骼中的一种主要细胞类型，目前仍不清楚。骨细胞凋亡与骨质疏松症和颌骨骨坏死等疾病有关，最近的证据表明，这种细胞类型在调节骨形成/矿化和调节磷酸盐稳态中起着重要作用。因此，了解这种细胞类型的功能将有助于开发骨质疏松症，骨软化症和其他代谢性骨骼疾病的治疗方法。

DESCRIPTION (provided by applicant): Osteocytes make up over 90% of the cells in bone. However, little is known about their function, due to difficulties in accessing these cells in vivo or isolating them for in vitro studies. Although the osteocyte has classically been viewed as an inactive cell, evidence is accumulating that these cells are responsive to mechanical loading and are able to modify their local environment, suggesting that they may be more active than previously thought. Using a transgenic mouse in which green fluorescent protein (GFP) is targeted to osteocytes under control of the dentin- matrix protein-1 (DMP1) promoter, we have performed time lapse dynamic imaging studies on living osteocytes in calvarial organ cultures. Surprisingly, these studies have revealed that, far from being a static cell, the osteocyte is highly dynamic. Osteocytes that were embedded within their lacunae expanded and contracted their cell bodies and extended and retracted their dendrites. Dendritic connections between osteocytes and with motile cells on the bone surface appeared to be transient, with connections being made and broken. These observations raise the possibility that dendrites, rather than being permanent intercellular connections, may be dynamic structures that can be altered in response to stimuli affecting osteocyte function. The central hypothesis for the proposed studies is that osteocytes within bone are highly dynamic cells and that their cell body and dendrite motions are critical to their function and to the embedding process. To test this hypothesis, multidisciplinary approaches will be used, including the use of transgenic mouse models for dynamic imaging of living osteocytes and osteoblasts, together with computational analysis of cell body and dendrite motions. In aim 1 we will use state-of-the-art imaging techniques to determine the dynamic properties of osteocytes and their dendrites in living bone explants. We will determine whether these change with age and/or in response to external stimuli known to affect osteocyte function, such as hypoxia and prostaglandin E2. The effects of inhibitors of cell motility and actin polymerization/ depolymerization on gap junctional signaling as an endpoint of osteocyte function will be determined. In aim 2 we will use a bone forming organ culture model to dynamically image osteoblasts embedding and differentiating into osteocytes. This will be done using calvaria from mice expressing DsRed in osteoblasts and GFP in osteocytes, driven by the Col1a1 and DMP1 promoters, respectively. Mineral deposition will also be imaged to determine how the dynamics of osteoblast embedding/differentiation are integrated with mineralization. The effects of inhibitors of cell motility and actin polymerization/depolymerization on embedding and mineralization will be determined. Successful completion of these exploratory studies will develop new and innovative models to facilitate research into osteocyte function, provide fundamental insights into the process of osteoblast to osteocyte transition and provide a new paradigm for viewing osteocyte dendrites as dynamic interactive structures. This will have major implications for our understanding the role of osteocytes in normal bone and in diseases, such as osteoporosis and osteomalacia. These studies will lay the foundation for an RO1 application in 1-2 years. This research is relevant to public health as it will provide highly novel insights into the function of the osteocyte, a major cell type in bone, which is still not well understood. Osteocyte apoptosis has been implicated in diseases such as osteoporosis and osteonecrosis of the jaw and recent evidence suggests that this cell type plays a major role in regulation of bone formation/mineralization and in regulation of phosphate homeostasis. Understanding the function of this cell type will therefore aid the development of treatments for osteoporosis, osteomalacia and other metabolic bone diseases.