REGULATION OF MATRIX GENE EXPRESSION IN CHONDROCYTES

软骨基质的修复缺陷是导致 骨关节炎（OA）和其他关节软骨功能丧失 关节炎 在过去，诱导和 维持人体软骨细胞的合成代谢活动受到阻碍 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， 确定的培养条件。 在这一建议中，一个新成立的 将使用温度敏感性关节软骨细胞系（tsT/AC 62 在细胞和分子水平上确定 在诱导和维持“适当的”合成模式方面， 软骨特异性胶原蛋白和其他基质蛋白。 是我们 细胞增殖和维持下调假说 3-在关键合成代谢因素存在下的三维环境 将允许软骨细胞表型和基质的表达 体外和体内沉积。 我们的目标是发展3- 三维培养模型，其中软骨细胞表型是 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具体目的：（1）确定 维持和增强永生化软骨细胞表型的因子 人软骨细胞体外培养模型。(2)识别分子 参与诱导和维持分化的机制 软骨细胞表型的直接分析的调控序列的 软骨特异性II型胶原基因（COL 2A 1）。(3)检查因素 影响体内软骨形成。 基因植入 在三维胶原蛋白基质中的工程化人软骨细胞， SCID小鼠将验证体外获得的结果，并测试 这些细胞参与真正的软骨修复的能力。 可用于研究的人软骨细胞培养模型的可用性 可重复地操作以进行确定的分化程序 将使我们能够更详细地研究 在体外和体内调节COL 2A 1表达。 一个 理解诱导和维持软骨细胞的关键信号 表型，但可能不存在于微环境中， 成人OA软骨细胞，最终将提供合理的策略， 用软骨细胞改善软骨修复的离体基因治疗 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.