REGULATION OF CARTILAGE REPAIR-SPECIFIC GENE EXPRESSION

描述(改编自申请人的摘要) 在骨关节炎(OA)中，有证据表明失去了同步性 在细胞外基质(ECM)分解代谢和合成代谢之间。我们有 观察到的ECM基因的数量和质量差异 比较正常软骨维护与软骨的表达 修理。在这项提议中，具体目标解决了中心假设 软骨细胞修复特异性基因的表达受特异性调控 涉及整合素的细胞-基质相互作用。在具体目标1中，我们将 确定连接蛋白快速上调的机制和 急性基质损伤后软骨修复过程中aggrecan基因的表达 被蛋白酶耗尽。将进行核子连续实验，以 评估连接蛋白和集聚蛋白基因的瞬时速率 高密度蛋白酶处理对转录的响应 软骨细胞培养。我们将检测连接蛋白和凝集素蛋白。 放线菌素D阻断软骨细胞转录的半衰期 将决定连接蛋白3‘UTR调控RNA序列 与细胞质蛋白(AUBF)相互作用可能授予mRNA 稳定状态。在具体目标2中，我们将定义 软骨细胞对细胞外基质改变的识别作用 基因表达发生了改变。我们将鉴定存在于牛身上的整合素 通过Northern杂交分析软骨细胞，并将确定 整合素亚单位mRNA在牛和人软骨中的分布 原位聚合酶链式反应/原位杂交。软骨细胞基因的变化 在用抑制剂治疗后将测量其表达 整合素-基质相互作用及与二氢细胞松弛素B的相互作用 微丝网络。在具体目标3中，我们将描述 修复基因在牛软骨中表达的细胞模式 实验操作后的外植体，并将这些关联 人骨关节炎软骨的研究结果。我们将使用原位聚合酶链式反应/原位 杂交决定修复特异性基因的细胞模式 耗竭的牛软骨和人骨关节炎软骨中的表达。 为了证实和扩展这些研究，我们还将使用免疫组织化学 软骨移植中修复特异性表位的定位技术 被酶处理耗尽的。这项研究将产生新的 可能允许对细胞基质进行治疗性操作的信息 刺激骨性关节炎软骨基质合成和修复的相互作用， 为骨性关节炎的病理生理干预提供了新的途径。

DESCRIPTION (Adapted from the Applicant's Abstract) In osteoarthritis (OA) , there is evidence for a loss of synchrony between extracellular matrix (ECM) catabolism and anabolism. We have observed quantitative as well as qualitative differences in ECM gene expression when comparing normal cartilage maintenance with cartilage repair. In this proposal, specific aims address the central hypothesis that chondrocyte repair-specific gene expression is regulated by specific cell-matrix interactions involving integrins. In specific aim 1 we will determine the mechanism for the rapid upregulation of link protein and aggrecan mRNA observed during cartilage repair following acute matrix depletion by proteases. Nuclear run-on experiments will be performed to assess the instantaneous rate of link protein and aggrecan gene transcription in response to protease treatment of high density chondrocyte cultures. We will measure link protein and aggrecan mRNA half-life by blocking chondrocyte transcription using actinomycin D. We will determine whether link protein 3' UTR regulatory RNA sequences interact with a cytoplasmic protein (AUBF) that may confer mRNA stabilization. In specific aim 2 , we will define the mechanism for chondrocyte recognition of extracellular matrix alterations leading to altered gene expression. We will identify integrins present on bovine chondrocytes by Northern blot analysis and will determine the distribution of integrin subunit mRNA in bovine and human cartilage by in-situ PCR/in-situ hybridization. Changes in chondrocyte gene expression will be measured following treatment with inhibitors of integrin-matrix interactions and with dihydrocytochalasin B to disrupt microfilament networks. In specific aim 3, we will characterize the cellular pattern of expression of repair genes in bovine cartilage explants following experimental manipulation, and will correlate these findings with human OA cartilage. We will use in-situ PCR/in-situ hybridization determine the cellular pattern of repair-specific gene expression in depleted bovine cartilage explants and human OA cartilage. To confirm and extend these studies, we will also use immunohistochemical techniques to localize repair-specific epitopes in cartilage explants depleted by enzymatic treatment. This research will yield new information that may permit therapeutic manipulation of cell-matrix interactions to stimulate matrix synthesis and repair of OA cartilage, and provide a novel approach to intervention in OA pathophysiology.