IN VITRO INITIATION OF BIOLOGICAL CALCIFICATION

生物钙化的体外引发

• 批准号: 2517436
• 负责人: ADELE L BOSKEY
• 金额: $ 18.76万
• 依托单位: HOSPITAL FOR SPECIAL SURGERY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-08-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2517436
• 关键词: X ray crystallography; blocking antibody; calcification; casein kinase; cell cycle; chondrocytes; collagen; electron microscopy; enzyme inhibitors; extracellular matrix proteins; gene expression; growth factor; infrared spectrometry; interferometry; normal ossification; osteoarthritis; phosphoprotein phosphatase; phosphorylation; posttranslational modifications; protein biosynthesis; protein structure; skeletal disorder chemotherapy; tissue /cell culture

DESCRIPTION (Adapted from the applicant's Abstract): The major objective of the proposed studies is the elucidation of the mechanism of cell controlled biomineralization. The differentiating chick limb-bud mesenchymal cell micromass culture system will be studied. The Principal Investigator proposes to utilize this culture system because of the economy and reproducibility of the system, the availability of markers, and her previous experience with these cultures. The Principal Investigator has already demonstrated that conclusions derived from this avian model are generally applicable to mineralization mechanisms in other species. Prior results showed that phosphorylation of matrix proteins is a key step in the mineralization process and that mineralization is actively controlled by cells, even after the first mineral crystals are deposited. In the proposed studies, the Principal Investigator and her collaborators will focus on the initial events in the mineralization process and on those events that control the cell mediated proliferation of mineral crystals. The Principal Investigator proposes that an understanding of these events is crucial for the development of techniques for promoting bone ingrowth, treating osteoarthritis and its complications, and treating developmental abnormalities. The Principal Investigator will study the mechanisms of cell mediated calcification using physico-chemical and physical chemical techniques, supplemented with techniques from cell and molecular biology. Three specific hypotheses will be tested with evaluations based on Fourier Transform infrared microscopy, x-ray diffraction, electron microscopy, and chemical analyses. The first hypothesis to be tested in Aim 1 is that appropriate cell mediated phosphoprotein phosphorylation and dephosphorylation regulate the site of initial mineral deposition, the rate and extent of mineral deposition, and the properties of the mineral crystals. Studies will include inhibition of casein kinase-like enzymes and phosphoprotein phosphatases as well as overexpression of the phosphoprotein(s) identified as important for control of mineralization. The second hypothesis to be tested in Aim 2 is that both the cellular production and the extracellular post translational processing of collagens regulates the site of initial mineral deposition, the rate and extent of mineral deposition and the mineral crystals' properties. Experiments will be performed by antibody blocking of specific collagen epitopes (i.e. immunoblocking) and by altering collagen cross-links. The third hypothesis to be tested in Aim 3 is that both mature cells and mature matrices are needed for initiation of calcification and progression of crystal growth. The maturation process will be varied by exposing the cultures to load (i.e. stretching), and to specific growth factors (OP-l, vitamin A, dexamethasone) which are known to accelerate chondrocyte maturation. Emphasis will be placed on defining the characteristics of the mineral formed and on defining if mineralization is associated with alterations in the degree of phosphorylation of phosphoproteins or if it is collagen type or structure specific.

描述(改编自申请人的摘要)：主要目标 提出的研究是对细胞控制机制的阐明。 生物矿化作用。分化中的雏鸡肢芽间充质细胞 将对微生物群体培养系统进行研究。首席调查员 建议利用这种文化系统，因为经济和 系统的重复性，标记的可用性，以及她以前的 体验这些文化。首席调查员已经 论证了从这一鸟类模型得出的结论通常是 适用于其他物种的矿化机制。之前的结果 研究表明，基质蛋白的磷酸化是 成矿过程和成矿作用受 细胞，即使在第一个矿物晶体沉积之后也是如此。在建议的 研究，首席调查员和她的合作者将专注于 成矿过程中的初始事件以及那些 控制细胞介导的矿物晶体的增殖。《校长》 调查人员提出，对这些事件的理解对于 促进骨长入、治疗的技术进展 骨性关节炎及其并发症及其治疗进展 异常现象。首席调查员将研究细胞的机制 物理化学和物理化学介导性钙化 技术，辅以细胞和分子生物学的技术。 三个具体的假设将通过基于傅立叶分析的评估进行检验 变换红外显微镜、x射线衍射、电子显微镜和 化学分析。在目标1中要检验的第一个假设是 适当的细胞介导的磷蛋白磷酸化和 去磷酸化调节初始矿物质沉积的位置，速率 和矿物沉积的程度，以及矿物的性质 水晶。研究将包括抑制酪蛋白激酶样酶和 磷酸化蛋白磷酸酶及其在细胞中的过度表达 磷蛋白(S)被认为是控制矿化的重要因素。 在目标2中要检验的第二个假设是，细胞 胶原蛋白的产生及其胞外翻译后加工 规定了初始矿物沉积的地点、速度和程度 矿物沉积和矿物晶体的性质。实验将会 通过抗体封闭特定的胶原表位(即 免疫阻断)和改变胶原交联物。第三个假设 在目标3中要测试的是成熟细胞和成熟基质都是 启动钙化和晶体生长进程所需的。 成熟过程将通过将培养物暴露在负载(即 拉伸)，以及特定的生长因子(OP-L、维生素A、地塞米松) 它们可以加速软骨细胞的成熟。重点将是 放在定义所形成的矿物的特征和定义上 如果矿化与矿化程度的变化有关 磷蛋白的磷酸化或是否为胶原型或结构 具体的。