GAP JUNCTIONS AND BONE CELL RESPONSE TO PHYSICAL SIGNALS

描述(改编自调查者摘要)：机械载荷 调节骨骼新陈代谢，但其发生机制尚不清楚 明白了。申请人建议将机械载荷传递到骨骼。 通过负载诱导的物理信号，如流体诱导剪切的细胞 压力。此外，FLOW刺激表型特征的表达 通过动员成骨细胞的机制向成骨细胞分化 细胞内钙与细胞间隙连接通讯(GJIC) 成骨细胞或成骨细胞。这个项目的长期目标是 检查应用于成骨细胞或成骨细胞的FLOW对 成骨细胞分化及其与细胞内钙和GJIC的关系 这一过程。此外，甲状旁腺激素(PTH)对 GJIC激活增强FLOW对成骨细胞的作用 将对活动进行检查。这些目标将通过 完成四个具体目标。目标1是量化流动对 细胞内钙动员与成骨细胞标志物的表达 人胎成骨细胞系(hFOB 1.19)和小鼠的分化 颅骨成骨细胞(MCOB)。目标2建议量化以下因素的影响 缝隙连接在hFOB 1.19和MCOB细胞中表达和功能的研究进展 成骨细胞分化标志物在这些细胞中的表达 带和不带功能缝隙连接。在目标3中，研究将审查 GJIC在成骨细胞流对其活性影响中的作用 未暴露于流动的成骨细胞。目标4建议进行研究以量化 甲状旁腺激素对成骨细胞或成骨细胞的流动影响 成骨细胞。细胞内钙浓度将通过以下方式定量 Fura-2微量荧光分光光度法。作为成骨细胞的标志物 分化，建议通过以下方法测定碱性磷酸酶的活性 分光光度法，骨钙素合成，通过免疫放射分析，和 I型胶原、骨桥蛋白和CBFA1mRNA的稳态水平 Time RT-PCR法。GJIC将通过染料转移技术进行评估。最近的 已建立的MLO-Y4细胞系将作为成骨细胞模型。HFOB 1.19和MLO-Y4细胞因药理作用而导致GJIC缺陷 阻滞剂、反义策略和抑制肽。分离的MCOB细胞 来自Cx43基因缺失的小鼠也将被利用。流体流动将应用于 平行板室和旋转盘室。这个项目的成果 将提供对骨细胞适应其功能的机制的洞察 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.