SHEAR STRESS AND CHONDROCYTE GENE EXPRESSION

虽然退行性关节疾病骨关节炎的确切病因尚不清楚，但疼痛和残疾的临床表现最常与不适当的机械负荷有关。正如手术中所见，关节软骨的局限性侵蚀最终导致关节功能的丧失，并且仍然是所有骨关节炎病例的最终共同途径，无论其原因如何。本研究的目的是确定机械负荷影响软骨基质合成和降解的细胞机制。在关节中，软骨细胞，软骨细胞，受到一系列复杂的应力和应变。我们的工作表明，培养的正常软骨细胞对施加间歇性静水压力或流体诱导剪应力的单变量机械刺激产生代谢反应。这里要检验的假设是，不同的细胞内信号通路是关节软骨对两种形式的机械刺激做出反应的基础。前炎性细胞因子白介素1在抑制软骨细胞外基质大分子合成和诱导软骨细胞降解酶合成中的作用已有基础知识。然而，在IL-1存在的情况下，机械负荷对关节软骨细胞降解酶表达的影响仍不清楚。这些特定的目的将量化切应力(SS)和间歇静水压力(IHP)对体外培养的人骨关节炎关节软骨细胞的影响，以：(1)检验IHP和SS差异性调控OA和正常软骨细胞细胞外基质大分子表达的假说；(2)检验IHP和SS调控IL-1β抑制OA和正常软骨细胞蛋白多糖和II型胶原合成的假说；(3)检验IHP和SS改变内源性MMPs和agrecanase在OA和正常软骨细胞表达的假说；(4)检验IHP和SS改变OA和正常软骨细胞MMPs和recanse表达的假说。预期的结果是，SS和IHP将显示出不同的能力来克服IL-1诱导的软骨细胞代谢从软骨维持向基质破坏的转变。这一研究结果将对骨科、风湿病和康复医学领域具有重要意义。这些技术将包括通过掺入放射性标记前体来分析蛋白多糖和胶原合成。RNA信号水平将通过Northern印迹和RT-PCR分析进行量化。细胞因子将通过生物测定和商业上可用的ELISA法进行量化。MMPs将通过酶谱和Western blotting进行鉴定，并通过

While the precise etiology of the degenerative joint disease, osteoarthritis, is unknown, clinical manifestations of pain and disability are most often associated with inappropriate mechanical loading. As evident at surgery, focal erosion of articular cartilage culminates in loss of joint function and remains the final common pathway in all cases of osteoarthritis irrespective of cause. The goal of this study is to determine the cellular mechanisms by which mechanical loading influences cartilage matrix synthesis and degradation. In the joint, cartilage cells, chondrocytes, are subject to a complex array of stresses and strains. Our work shows that normal chondrocytes in culture react metabolically to univariate mechanical stimulation applied either as intermittent hydrostatic pressure or as fluid- induced shear stress. The hypothesis to be tested here is that distinct intracellular signaling pathways underlie the articular cartilage response to the two forms of mechanical stimulation. Fundamental knowledge exists regarding the effects of the proinflammatory cytokine, interleukin-1, on inhibition of cartilage extracellular matrix macromolecule synthesis and induction of cartilage degrading enzyme synthesis by chondrocytes. However, the effects of mechanical loading on the expression of articular chondrocyte degradative enzymes in the presence of IL-1 remain unclear. The specific aims will quantify effects of shear stress (SS) and intermittent hydrostatic pressure (IHP) on human osteoarthritic articular chondrocytes in vitro to: (1) Test the hypothesis that IHP and SS differentially modulate extracellular matrix macromolecule expression in OA versus normal chondrocytes; (2) Test the hypothesis that IHP and SS modulate IL-1beta induced inhibition of proteoglycan and type II collagen synthesis in OA and normal chondrocytes; (3) Test the hypothesis that IHP and SS alter endogenous MMPs and aggrecanase expression in OA and normal chondrocytes; (4) Test the hypothesis that IHP and SS alter IL-1beta induced expression of MMPs and aggrecanse in OA and normal chondrocytes. The expected result is that SS and IHP will show dissimilar capacities to overcome the IL-1 induced shift of chondrocyte metabolism from cartilage maintenance to matrix destruction. The results of this study will be of importance to the fields of orthopaedics, rheumatology and rehabilitation medicine. The techniques will involve analysis of proteoglycan and collagen synthesis by incorporation of radiolabeled precursors. mRNA signal levels will be quantified by Northern blotting and RT-PCR analysis. Cytokines will be quantified by bioassays and commercially available ELISA. MMPs will be identified by zymography and Western blotting and quantified by