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通常与不适当的机械负荷有关。 如手术中所示，关节软骨局灶性侵蚀最终导致关节功能丧失，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，正常的软骨细胞在培养代谢反应单变量机械刺激施加作为间歇静水压力或流体诱导的剪切应力。 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的反应的基础。关于促炎细胞因子白细胞介素-1对软骨细胞外基质大分子合成的抑制和软骨细胞对软骨降解酶合成的诱导的作用存在基础知识。 然而，在IL-1存在下，机械负荷对关节软骨细胞降解酶表达的影响仍不清楚。 本研究的具体目的是量化剪切应力（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对OA与正常软骨细胞中细胞外基质大分子表达的差异调节的假设;（2）验证IHP和SS调节IL-1 β诱导的OA和正常软骨细胞蛋白多糖和II型胶原合成抑制的假设;（3）检验IHP和SS改变OA和正常软骨细胞中内源性MMPs和聚集蛋白聚糖酶表达的假设;（4）检验IHP和SS改变OA和正常软骨细胞中IL-1 β诱导的MMPs和聚集蛋白聚糖酶表达的假设。 预期的结果是，SS和IHP将显示出不同的能力，以克服IL-1诱导的软骨细胞代谢从软骨维持到基质破坏的转变。 本研究结果对骨科、风湿病学和康复医学等领域具有重要意义。 该技术将涉及通过掺入放射性标记的前体来分析蛋白聚糖和胶原蛋白合成。 通过北方印迹和RT-PCR分析定量mRNA信号水平。 将通过生物测定和市售ELISA定量细胞因子。MMP将通过酶谱法和蛋白质印迹法鉴定，并通过ELISA定量。

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