Mechanical Loading and Matrix Metalloproteinase

描述（由申请人提供）：本提案的长期目标是开发一种高分辨率力加载器，用于对结缔组织施加精确载荷，并阐明机械应力和应变对类风湿关节的作用。胶原蛋白是软结缔组织中的主要成分，胶原蛋白的周转至少部分受到蛋白水解酶家族的调节，基质金属蛋白酶（MMPs）被认为是骨和软结缔组织中有影响力的蛋白酶组。在分析机械应力和应变在基质降解中的作用时，精确控制机械载荷是很重要的，因为MMPs的表达和活性在很大程度上取决于剪切和应变的强度、梯度和持续时间。在这个修订后的为期两年的生物工程研究项目中，一个具体的目标是设计和制造一个最先进的压电器件，可以与倒置荧光和光学显微镜一起使用。生命科学家将能够在机械刺激下实时表征细胞形态和分子动力学。

DESCRIPTION (provided by applicant): The long-term objectives of this proposal are to develop a high-resolution force loader for applying precise loads to connective tissues, and to elucidate the role of mechanical stress and strain to rheumatoid joints. A turnover of collagens, predominant components in soft connective tissues, is at least in part regulated by families of proteolytic enzymes, and the matrix metalloproteinases (MMPs) have been considered as the influential group of proteinases in bone and soft connective tissues. In analyzing the role of mechanical stress and strain in matrix degradation, precise control of mechanical loads is important since the expression and the activities of MMPs depends heavily upon intensities, gradients, and duration of shear and strain. In this revised 2-year bioengineering research project, a specific aim is to design and fabricate a state-of-the-art piezo-electric device that can be utilized with inverted fluorescence and optical microscopy. Life scientists will be able to characterize cellular morphology and molecular dynamics in real-time under mechanical stimuli. The proposed piezo-electric device will be able to apply precise deformation of 100-500 um to cultured cells at 0.1-100 Hz sinusoidal or impulsive waveforms in varying directions. The device will be validated by reproducing the previous results showing that gentle cyclic stain alters the expression and the activities of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in human synovial cells. In determining the expression and activities of MMPs and TIMPs, we will use RT-PCR, Western blotting, zymography as well as in vitro and in situ fibril degradation assays. The proposed project will contribute to understanding effects of mechanical stimuli on cellular morphology as well as molecular activities. The device can be used to address fundamental questions about how mechanical forces affect cells in real time as well as to elucidate the mechanisms of inflammation and degradation of connective tissue under mechanical stimuli.