STRETCH ACTIVATED CHANNELS IN CHONDROCYTES

描述（改编自申请人的摘要）：尽管有密集的 研究工作，很少有人知道细胞机制 潜在的骨关节炎。 随着老年人口的不断增加， 受关节炎折磨的人的数量将增加， 迫切需要深入了解这种昂贵的病理生理学， 疾病 研究人员认为，更好地了解 软骨细胞中的生物物理信号转导是 了解骨关节炎。 他们建议将机械化学 在分离的软骨细胞中响应于流体流动的信号转导 从牛关节软骨中提取 他们的核心假设是， 软骨细胞中的生物物理信号转导至少部分被定义为 通过激活胞质Ca 2+的牵张激活（SA）通道 动员。 他们的长期目标是表征流体流动效应 对胞浆Ca 2+和蛋白多糖合成的影响以及SA通道的作用 把这两个答案联系起来。 这些目标将通过 实现四个具体目标。 1)量化，在真实的时间， 骨关节软骨细胞（BAC）暴露于 稳定和振荡流体流动; 2）检查膜拉伸诱导 通道活性BAC; 3）定量暴露于流体流动的BAC中的[Ca 2 +]i， 调节特定信号传导的因素的存在和不存在 4）定量BAC中聚集蛋白聚糖mRNA和蛋白聚糖的合成 暴露的流体流动。 将分离BAC，仅在传代培养后使用。 II型胶原 用间接免疫荧光和I型和 通过核酸酶保护试验检测II型胶原mRNA。 流体流动对 [Ca2+]i将通过显微荧光分光光度法定量。 SA通道功能 并通过膜片钳电生理学定量表达， 核酸酶保护测定。 聚集蛋白聚糖mRNA表达和 响应于流体流动的蛋白聚糖合成将通过 核酸酶保护测定和35 S-硫酸盐掺入。 这些实验 将在流体的特定抑制剂的存在下重复 流动诱导的胞质CA 2+动员。 该项目的成果将 深入了解生物物理信号调节 软骨细胞代谢

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.