STRETCH ACTIVATED CHANNELS IN CHONDROCYTES

伸展软骨细胞中的激活通道

• 批准号: 2457615
• 负责人: Henry J Donahue
• 金额: $ 21.11万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2457615
• 关键词: RNase protection assay; aging; articular cartilage; biological signal transduction; calcium channel blockers; calcium flux; calcium indicator; cell membrane; chondrocytes; chondroitin sulfates; collagen; cow; fluid flow; fluorescent dye /probe; immunofluorescence technique; membrane activity; membrane channels; membrane potentials; oscillography; osteoarthritis; proteoglycan; voltage /patch clamp

DESCRIPTION (Adapted from the Applicant's Abstract): Despite an intensive research effort, very little is known about the cellular mechanism underlying osteoarthritis. As the aged population continues to increase, the number of individuals afflicted with arthritis will increase emphasizing the critical need for insights into the pathophysiology of this costly disease. The investigators believe that a better understanding of biophysical signal transduction in chondrocytes is a critical first step in understanding osteoarthritis. They propose to characterize mechano-chemical signal transduction, in response to fluid flow, in chondrocytes isolated from bovine articular cartilage. Their central hypothesis is that biophysical signal transduction in chondrocytes is at least partly defined by stretch activated (SA) channels which activate cytosolic Ca2+ mobilization. Their long term goals are to characterize fluid flow effects on cytosolic Ca2+ and proteoglycan synthesis and the role SA channels play in linking these two responses. These goals will be accomplished through the completion of four specific aims. 1) Quantify, in real time, the cytosolic Ca2+ concentration of bone articular chondrocytes (BAC) exposed to steady and oscillatory fluid flow; 2) Examine membrane stretch-induced channel activity BAC; 3) Quantify [Ca2+]i in BAC exposed to fluid flow in the presence and absence of factors which regulate specific signalling pathways; and 4) Quantify aggrecan mRNA and proteoglycan synthesis in BAC exposed fluid flow. BAC will be isolated and used after only subculture. Type II collagen expression will be monitored with indirect immunofluorescence and type I and II collagen mRNA by nuclease protection assays. Fluid flow effects on [Ca2+]i will be quantified by microspectrofluometry. SA channel function and expression will be quantified by patch clamp electrophysiology and nuclease protection assays, respectively. Aggrecan mRNA expression and proteoglycan synthesis, in response to fluid flow, will be quantified by nuclease protection assays and 35S-sulfate incorporation. These experiments will be repeated in the presence of specific inhibitors of fluid flow-induced cytosolic CA2+ mobilization. The results of this project will provide insights into the mechanism by which biophysical signals regulate chondrocyte metabolism.

描述(改编自申请人的摘要)：尽管 研究工作，对细胞机制知之甚少 潜在的骨性关节炎。随着老龄人口的持续增加， 患关节炎的人数将会增加，强调 迫切需要深入了解这一代价高昂的 疾病。调查人员认为，更好地了解 软骨细胞的生物物理信号转导是关键的第一步 了解骨关节炎。他们建议将机械力化学描述为 在分离的软骨细胞中响应流体流动的信号转导 取自牛关节软骨。他们的中心假设是 软骨细胞中的生物物理信号转导至少有部分定义 通过拉伸激活(SA)通道激活胞浆钙离子 动员。他们的长期目标是描述流体流动的影响。 细胞内钙离子和蛋白多糖的合成及SA通道的作用 将这两种反应联系起来。这些目标将通过以下方式实现 四个具体目标的完成。1)实时量化 染毒骨关节软骨细胞胞浆钙离子浓度的变化 稳定和振荡的流体流动；2)检查薄膜拉伸诱导 BAC的通道活性；3)BAC中[Ca~(2+)]i的定量 调节特定信号的因素的存在和缺失 4)定量测定BAC中aggrecan mRNA和蛋白多糖的合成 暴露的液体流动。 BAC只有在继代培养后才会被分离和使用。II型胶原 将用间接免疫荧光和I型和 用核酸酶保护法测定II型胶原基因的表达。流体流动的影响 [Ca~(2+)]i将用微量荧光分光光度法定量。SA通道功能 和表达将通过膜片钳电生理学和 核酸酶保护实验。聚集素蛋白聚糖基因的表达及其意义 蛋白多糖的合成，作为对流体流动的响应，将通过 核酸酶保护试验和35S-硫酸盐掺入试验。这些实验 会在特定的体液抑制剂存在的情况下重复 流动诱导的胞浆内钙离子动员。这个项目的结果将是 提供对生物物理信号调节机制的见解 软骨细胞新陈代谢。