REGULATION OF MATRIX GENE EXPRESSION IN CHONDROCYTES

软骨基质的修复缺陷是导致软骨损伤的主要原因 骨关节炎等的关节软骨功能丧失 关节炎。在过去，对诱导和 人类软骨细胞维持合成代谢活动受到阻碍 由于缺乏合适的软骨细胞培养系统。最近，我们 报道了永生化人类的发展和特征 表达软骨特异性基质蛋白的软骨细胞系 定义的培养条件。在这项提案中，一个新成立的 将使用温度敏感型关节软骨细胞系(TST/AC62) 在细胞和分子水平上定义所涉及的机制 在诱导和维持“适当的”合成模式中 软骨特有的胶原蛋白和其他基质蛋白。这是我们的 假设细胞增殖和维持的下调 存在临界合成代谢因子的三维环境 将允许软骨细胞表型和基质的表达 体外和体内沉积。我们的方法将是开发3- 软骨细胞表型为 在体外增强和维持并确定关键因素 控制软骨特异性基因表达和基质合成 在体外和体内通过以下特定目的：(1)测定 永生化软骨细胞表型维持和增强的因素 人软骨细胞体外培养模型。(2)分子鉴定 诱导和维持分化的机制 直接分析软骨细胞表型的调控序列 软骨特异性II型胶原基因(COL2A1)。(三)考察因素 影响体内软骨的形成。遗传性植入 人软骨细胞在三维胶原基质中的工程化 SCID小鼠将验证在体外获得的结果，并测试 这些细胞参与真正的软骨修复的能力。 人软骨细胞培养模型的可用性 可重现地被操纵以经历特定的区分程序 将使我们能够更详细地研究分子机制 在体外和体内调节COL2A1的表达。一个 对诱导和维持软骨细胞的关键信号的理解 表型，但这可能不存在于 成人骨关节炎软骨细胞，最终将提供合理的治疗策略 软骨细胞促进软骨修复的体外基因治疗 移植方法。

Defective repair of cartilage matrix is a major feature accounting for loss of function of articular cartilage in osteoarthritis (OA) and other arthritides. In the past, definition of the mechanisms that induce and maintain anabolic activities in human chondrocytes have been hampered by the lack of suitable chondrocyte culture systems. Recently, we reported the development and characterization of immortalized human chondrocyte lines that express cartilage-specific matrix proteins under defined culture conditions. In this proposal, a newly established temperature-sensitive articular chondrocyte line (tsT/AC62) will be used to define at the cellular and molecular level the mechanisms involved in the induction and maintenance of "appropriate" patterns of synthesis of cartilage-specific collagen, and other matrix proteins. It is our hypothesis that down-regulation of proliferation and maintenance in a 3-dimensional environment in the presence of critical anabolic factors will permit expression of the chondrocyte phenotype and matrix deposition in vitro and in vivo. Our approach will be to develop 3- dimensional culture models in which the chondrocyte phenotype is enhanced and maintained in vitro and identify critical factors controlling cartilage-specific gene expression and matrix synthesis both in vitro and in vivo by the following specific aims: (1) Determine factors that maintain and enhance chondrocyte phenotype in immortalized human chondrocyte culture models in vitro. (2) Identify molecular mechanisms involved in induction and maintenance of differentiated chondrocyte phenotype by direct analysis of regulatory sequences of the cartilage-specific type II collagen gene (COL2A1). (3) Examine factors influencing cartilage formation in vivo. Implantation of genetically engineered human chondrocytes in a three-dimensional collagen matrix in SCID mice will validate the results obtained in vitro and test the capacity of these cells to participate in authentic cartilage repair. The availability of human chondrocyte culture models that can be manipulated reproducibly to undergo defined programs of differentiation will allow us to examine in greater detail the molecular mechanisms regulating COL2A1 expression both in vitro and in vivo. An understanding of critical signals that induce and maintain chondrocyte phenotype, but that may not be present in the microenvironment of the adult OA chondrocyte, will eventually provide rational strategies for ex vivo gene therapy for improving cartilage repair with chondrocyte transplantation approaches.