CHONDROCYTE GENE EXPRESSION DURING CARTILAGE REPAIR

细胞外基质生物合成和周转的调控 关节软骨中的组件必须涉及到 软骨细胞及其直接的细胞周围环境。在骨关节炎中 来自人类的软骨以及疾病的动物模型，在那里 发生蛋白水解酶介导的细胞外基质分子丢失。这 可能进而导致破坏正常粘合剂之间的相互作用 软骨细胞及其直接的细胞周围环境。失去正常状态 细胞-基质相互作用可能深刻影响软骨细胞基因 表达，这可能部分解释了软骨明显不能 有效修复蛋白水解酶介导的基质损伤。自关节 软骨新陈代谢不容易在体内研究，外植体培养 已经被用作模型系统来研究 软骨细胞对伤害性刺激的反应。我们建议检验这个假设 去基质软骨中软骨细胞的生物合成活性不同于 这发生在正常的软骨中。我们将定义软骨细胞 对矩阵更改的响应受 细胞外基质探针的可用性。初步努力已经 牛LINK特异性探针的分离与鉴定 蛋白质和聚集素，并正在努力产生探针 其他主要的细胞外基质成分。软骨的移植将会是 作为一个模型系统来研究软骨细胞对 通过外源添加的蛋白酶消耗细胞外基质，以 IL-1诱导的基质耗竭刺激内源性蛋白酶活性 在基质耗竭的兔骨关节炎软骨中。基因表达水平 细胞外基质蛋白的转录本将由 软骨细胞RNA的Northern印迹分析。使用cDNA文库 减法策略将努力鉴定表型 正常人和修复者之间基因表达的差异 软骨细胞来自基质耗尽的软骨。库减法将 允许识别细胞内和细胞外的序列 软骨细胞中表达受调控的基质软骨细胞蛋白 只根据表型的变化参与软骨修复， 不需要蛋白质或DNA序列的先验知识。

Regulation of the biosynthesis and turnover of extracellular matrix components in articular cartilage must involve an interplay between the chondrocyte and its immediate pericellular environment. In osteoarthritic cartilage from humans as well as in animal models of the disease, there occurs a protease mediated loss of extracellular matrix molecules. This may in turn lead to a disruption in normal adhesive interactions between the chondrocyte and its immediate pericellular environment. Loss of normal cell-matrix interactions could profoundly influence chondrocyte gene expression, and may explain in part the apparent inability of cartilage to effectively repair protease-mediated matrix damage. Since articular cartilage metabolism cannot easily be studied in vivo, explant cultures have been used as model systems for investigating the response of chondrocytes to injurious stimuli. We propose to test the hypothesis that chondrocyte biosynthetic activity in matrix-depleted cartilage differs from that occurring in normal cartilage. We will define the chondrocyte response to matrix alterations at a level of resolution limited by the availability of extracellular matrix probes. Preliminary efforts have involved isolation and characterization of bovine specific probes for link protein and aggrecan and efforts are in progress to generate probes for other major extracellular matrix components. Explants of cartilage will be used as a model system to study the response of the chondrocyte to depletion of the extracellular matrix by exogenously added proteases, to matrix depletion induced by IL-1 stimulated endogenous protease activity and in matrix-depleted osteoarthritic rabbit cartilage. Levels of mRNA transcripts for extracellular matrix proteins will be determined by Northern blot analysis of chondrocyte RNA. Using a cDNA library subtraction strategy an effort will be made to identify phenotypic differences in gene expression between the normal and the reparative chondrocyte from matrix-depleted cartilage. Library subtraction will permit the identification of sequences for intracellular and extracellular matrix chondrocyte proteins whose expression is modulated in chondrocytes involved in cartilage repair based only upon changes in phenotype, requiring no previous knowledge of protein or DNA sequences.