GAP JUNCTIONS AND BONE CELL RESPONSE TO PHYSICAL SIGNALS

描述（改编自研究者摘要）：机械载荷 调节骨代谢，但其发生的机制很差 明白申请人提出将机械载荷传递到骨 细胞通过负载引起的物理信号，如流体流动引起的剪切 应力此外，流动刺激表型特征的表达 分化成骨细胞通过一种机制，涉及动员 胞浆钙离子和间隙连接细胞间通讯（GJIC）， 成骨细胞或骨细胞。该项目的长期目标是 检查应用于骨细胞或成骨细胞的流动的效果， 成骨细胞分化和细胞内钙离子和GJIC在成骨细胞分化中的作用 这个过程此外，甲状旁腺激素（PTH）的能力， 通过激活GJIC，加强流动对成骨细胞的影响， 活动将被审查。这些目标将通过 实现四个具体目标。目标1是量化流量对 成骨细胞标志物的表达 在人胎儿成骨细胞系（hFOB 1. 19)和鼠标 颅骨成骨细胞（MCOB）。目标2建议量化 流动对hFOB 1中间隙连接表达和功能的影响。19和MCOB细胞， 在这些细胞中成骨细胞分化标志物的表达 有和没有功能性间隙连接。在目标3中，研究将审查 GJIC在施加于骨细胞的流动对骨细胞活性的影响中的作用 成骨细胞不暴露于流动。目标4建议进行研究， 在存在PTH的情况下对成骨细胞或成骨细胞的流动的影响 骨细胞细胞溶质钙浓度将通过 用Fura-2进行显微荧光分光光度测定。作为成骨细胞 分化，研究提出了测定碱性磷酸酶活性， 分光光度法，骨钙素合成，免疫放射测定，和 I型胶原、骨桥蛋白和CBFA 1 mRNA的稳态水平，通过真实的 时间RT-PCR。将通过染料转移技术评估GJIC。最近 建立的MLO-Y 4细胞系将用作骨细胞模型。hFOB 1. 19和MLO-Y 4细胞将通过药理学方法使GJIC缺陷。 阻断剂、反义策略和抑制肽。分离的MCOB细胞 也将利用来自Cx43缺失小鼠的细胞。流体流动将应用于 平行板室和旋转盘室。该项目的结果 将提供深入了解骨细胞适应其 细胞外环境

DESCRIPTION (adapted from the Investigator's abstract): Mechanical loads regulate bone metabolism but the mechanism by which this occurs is poorly understood. The applicant proposes that mechanical load is transduced to bone cells via load-induced physical signals such as fluid-flow-induced shear stress. Furthermore, flow stimulates expression of phenotypic characteristics of differentiating osteoblastic cells via a mechanism involving mobilization of cytosolic calcium and gap junctional intercellular communication (GJIC) in osteoblastic or osteocytic cells. The long-term goals of this project are to examine the effect of flow, applied to osteocytic or osteoblastic cells, on osteoblastic differentiation and the roles cytosolic calcium and GJIC play in this process. Additionally, the ability of parathyroid hormone (PTH) to potentiate, through activation of GJIC, the effect of flow on osteoblastic activity will be examined. These goals will be accomplished through the completion of four specific aims. Aim 1 is to quantify the effect of flow on cytosolic calcium mobilization and expression of markers of osteoblastic differentiation in a human fetal osteoblastic cell line (hFOB 1. 19) and mouse calvarial osteoblastic cells (MCOB). Aim 2 proposes to quantify the effect of flow on gap junction expression and function in hFOB 1. 19 and MCOB cells and on expression of markers of osteoblastic differentiation in these same cells with and without functional gap junctions. In Aim 3, studies will examine the role of GJIC in the effect of flow applied to osteocytic cells on activity of osteoblastic cells not exposed to flow. Aim 4 proposes studies to quantify the effect of flow in the presence of PTH applied to either osteoblastic or osteocytic cells. Cytosolic calcium concentration will be quantified by microspectrofluorometry with fura-2. As markers of osteoblastic differentiation, studies are proposed to assay alkaline phosphatase activity by spectrophotometry, osteocalcin synthesis, by immunoradiometric assay, and steady-state levels of type I collagen, osteopontin and CBFA1 mRNA, by real time RT-PCR. GJIC will be assessed by dye transfer techniques. The recently established MLO-Y4 cell line will be utilized as an osteocytic cell model. hFOB 1. 19 and MLO-Y4 cells will be rendered GJIC deficient by pharmacological blockers, antisense strategies and inhibiting peptides. MCOB cells isolated from Cx43 null mice will also be utilized. Fluid flow will be applied in a parallel plate chamber and rotating disk chamber. The results of this project will provide insights into the mechanisms by which bone cells adapt to their extracellular environment.