CELLULAR BASIS OF CRANIOFACIAL BONE DISORDERS

颅面骨疾病的细胞基础

• 批准号: 6379723
• 负责人: Philip A Osdoby
• 金额: $ 21.71万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-30 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6379723
• 关键词: RNase protection assay; acid phosphatase; biological signal transduction; bone disorder; cell cell interaction; cell differentiation; cell growth regulation; chickens; congenital oral /facial /cranial defect; cytokine receptors; enzyme linked immunosorbent assay; gene targeting; genetically modified animals; human tissue; immunocytochemistry; in situ hybridization; interleukin 8; laboratory mouse; monoclonal antibody; neutralizing antibody; osteoclasts; osteogenesis; pathologic bone resorption; physiologic bone resorption; polymerase chain reaction

Normal bone development and remodeling require complex humoral, cell- cell, and cell-matrix interactions. The integration of humoral and local signals is necessary to permit proper development, maintain mineral homeostasis, insure mechanical strength, and support haematopoeisis. There is now good evidence that osteoblasts can orchestrate osteoclast development and modulate osteoclast activity via paracrine signals. Information is just beginning to emerge that osteoclasts themselves synthesize and release factors that may influence bone remodeling. We therefore hypothesize that osteoclast-derived signals may function as autocrine effectors of osteoclast development and activity and therefore influence normal and pathological osteoclastogenesis and osteoclast-mediated bone resorption. We have shown that osteoclasts produce small proinflammatory molecules known as chemokines, such as IL-8 and GRO alpha and that such molecules can act to modulate osteoclast precursor recruitment, development, and activity. Therefore, to further investigate and define osteoclast autocrine regulation we propose the following specific aims: 1) Identify a profile of chemokines produced by human and mouse OC and determine which exhibit autocrine effects on OC function (bone resorption, motility, free radical production, TRAP, cathepsin K, and carbonic anhydrase II expression). The regulation of this subset of chemokines by select known modulators of OC function will be analyzed. Included here will be studies employing selective chemokine and chemokine receptor antagonists, chemokine neutralizing antibodies, and mouse OC-like cells formed in vitro from bone marrow obtained from IL-8 receptor knockout mice. 2) Identify and characterize the profile of chemokine receptors expressed on OC as a function of OC physiology, and pathophysiology. As part of this aim we will begin to elucidate the intracellular signal transduction pathways involved in chemokine modulation of OC activity. 3) Examine the potential role of chemokines in OC precursor recruitment and differentiation in vitro, in vivo, and in ovo. 4. Examine spatial and temporal aspects of chemokine and chemokine receptor expression in normal and pathological bone tissue by in situ hybridization and immunohistochemistry. Included here are mouse IL-8 receptor knockout studies. All of the above studies will use a combination of in vivo and in vitro approaches, and model systems including the mouse calvarial injection model for histomorphometric studies, human tissue sections for in situ hybridization and immunohistochemical analysis, isolated human and avian Ocs, human Oc-like cells, the mouse Oc-like cell developmental model, and cells obtained from an IL-8 receptor knockout mouse. Oc- chemokine production, mRNA steady state levels and regulation will be assessed by RT-PCP, RNAse protection assay, chemokine ELISA, and in situ hybridization techniques. Osteoclast development and activity will be evaluated based on functional, biochemical and molecular markers of the osteoclast phenotype, including bone resorption, osteoclast antigen expression tartarate-resistant acid phosphatase activity, and calcitonin receptor levels. Such studies are anticipated to reveal new aspects of normal bone remodeling mechanisms such as tooth eruption and have potential to lend insight into skeletal pathologies such as periodontal disease, implant loosening, osteoarthritis, other inflammatory skeletal disorders, and osteoporosis.

正常的骨骼发育和重塑需要复杂的体液，细胞- 细胞和细胞-基质的相互作用。幽默与幽默的融合 当地的信号是必要的，以允许适当的发展，维护 矿物质平衡，确保机械强度和支撑力 造血术。现在有很好的证据表明成骨细胞可以 协调破骨细胞发育和调节破骨细胞活动 旁分泌信号。刚刚开始出现的信息是 破骨细胞自身合成和释放可能影响 骨骼重塑。因此我们假设破骨细胞起源于 信号可能作为破骨细胞发育的自分泌效应器 和活动，从而影响正常和病理性 破骨细胞生成和破骨细胞介导骨吸收。我们有 研究表明，破骨细胞会产生小的促炎分子 趋化因子，如IL-8和Groα，以及这些分子可以发挥作用 调节破骨细胞前体的募集、发育和活性。 因此，进一步研究和确定破骨细胞自分泌 法规我们提出了以下具体目标：1)确定配置文件 人类和小鼠OC产生的趋化因子，并确定哪个展品 自分泌对OC功能(骨吸收、动力、游离)的影响 自由基产生、TRAP、组织蛋白酶K和碳酸氢酶II 表达)。SELECT对趋化因子亚群的调节 将分析已知的OC函数的调制器。此处将包含以下内容 应用选择性趋化因子和趋化因子受体的研究 拮抗剂、趋化因子中和抗体和小鼠OC样细胞 从IL-8受体基因敲除获得的骨髓在体外形成 老鼠。2)鉴定和鉴定趋化因子受体的特性 表达在OC上，作为OC生理学和病理生理学的函数。 作为这个目标的一部分，我们将开始阐明细胞内的信号 参与趋化因子调控OC活性的信号转导通路。 3)研究趋化因子在OC前体募集中的潜在作用 以及在体外、体内和卵子中的分化。4.审视空间 趋化因子和趋化因子受体表达的时间特征 正常和病理性骨组织的原位杂交和 免疫组织化学。这里包括小鼠IL-8受体基因敲除 学习。所有上述研究都将使用体内和 体外方法和包括小鼠颅骨的模型系统 组织形态计量学研究的注射模型，人体组织切片 原位杂交和免疫组织化学分析分离的人 和禽类OCS、人Oc样细胞、小鼠Oc样细胞发育 模型，以及从IL-8受体基因敲除小鼠获得的细胞。业主立案法团 趋化因子的产生、mRNA的稳定水平及其调控 采用RT-PCP、核糖核酸酶保护试验、趋化因子酶联免疫吸附试验和原位检测 杂交技术。破骨细胞的发育和活性将 根据功能、生化和分子标记进行评估 破骨细胞表型，包括骨吸收、破骨细胞抗原 抗酒石酸酸性磷酸酶活性和降钙素的表达 受体水平。这类研究预计将揭示出 正常的骨改建机制，如牙齿萌出和HAD 有可能深入了解骨骼病理，如牙周 疾病、种植体松动、骨关节炎、其他骨骼炎症 紊乱和骨质疏松。