STRETCH ACTIVATED CHANNELS IN CHONDROCYTES

描述（改编自申请人的摘要）：尽管密集 研究工作，但对细胞机制知之甚少 潜在的骨关节炎。 随着老年人口的不断增加， 患有关节炎的人数将会增加 迫切需要深入了解这种昂贵的病理生理学 疾病。 调查人员认为，可以更好地了解 软骨细胞中的生物物理信号转导是关键的第一步 了解骨关节炎。 他们建议表征机械化学 分离的软骨细胞中响应流体流动的信号转导 来自牛关节软骨。 他们的中心假设是 软骨细胞中的生物物理信号转导至少部分被定义 通过激活胞质 Ca2 的拉伸激活 (SA) 通道 动员。 他们的长期目标是表征流体流动效应 SA通道对细胞质Ca2和蛋白多糖合成的影响及作用 将这两个响应联系起来。 这些目标将通过 完成四项具体目标。 1) 实时量化 骨关节软骨细胞 (BAC) 的胞浆 Ca2 浓度 稳定和振荡的流体流动； 2) 检查膜拉伸引起的 通道活动BAC； 3) 定量暴露于流体流动的 BAC 中的 [Ca2]i 调节特定信号传导的因素是否存在 途径； 4) 定量 BAC 中聚集蛋白聚糖 mRNA 和蛋白聚糖合成 暴露的流体流动。 仅继代培养后即可分离并使用 BAC。 II型胶原蛋白 将通过间接免疫荧光和 I 型和 通过核酸酶保护测定检测 II 胶原蛋白 mRNA。 流体流动的影响 [Ca2]i 将通过显微分光荧光测定法进行定量。 SA通道功能 表达将通过膜片钳电生理学进行量化 分别进行核酸酶保护测定。 聚集蛋白聚糖 mRNA 表达和 响应于流体流动的蛋白多糖合成将通过以下方式进行量化 核酸酶保护测定和 35S-硫酸盐掺入。 这些实验 将在存在特定液体抑制剂的情况下重复 流动诱导的胞浆 CA2 动员。 该项目的成果将 提供对生物物理信号调节机制的见解 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.