Otero Miguel, Favero Marta, Dragomir Cecilia, Hachem Karim El, Hashimoto Ko, Plumb Darren A, Goldring Mary B
Laboratory for Cartilage Biology, The Hospital for Special Surgery, Weill Cornell Medical College, New York, NY, USA.
Methods Mol Biol. 2012;806:301-36. doi: 10.1007/978-1-61779-367-7_21.
The human adult articular chondrocyte is a unique cell type that has reached a fully differentiated state as an end point of development. Within the cartilage matrix, chondrocytes are normally quiescent and maintain the matrix constituents in a low-turnover state of equilibrium. Isolated chondrocytes in culture have provided useful models to study cellular responses to alterations in the environment such as those occurring in different forms of arthritis. However, expansion of primary chondrocytes in monolayer culture results in the loss of phenotype, particularly if high cell density is not maintained. This chapter describes strategies for maintaining or restoring differentiated phenotype by culture in suspension, gels, or scaffolds. Techniques for assessing phenotype involving primarily the analysis of synthesis of cartilage-specific matrix proteins as well as the corresponding mRNAs are also described. Approaches for studying gene regulation, including transfection of promoter-driven reporter genes with expression vectors for transcriptional and signaling regulators, chromatin immunoprecipitation, and DNA methylation are also described.
成年人类关节软骨细胞是一种独特的细胞类型，它已达到完全分化状态，作为发育的终点。在软骨基质中，软骨细胞通常处于静止状态，并使基质成分维持在低周转率的平衡状态。培养中的分离软骨细胞为研究细胞对环境变化（如不同形式关节炎中发生的变化）的反应提供了有用的模型。然而，原代软骨细胞在单层培养中的扩增会导致表型丧失，特别是如果不维持高细胞密度的话。本章描述了通过在悬浮液、凝胶或支架中培养来维持或恢复分化表型的策略。还描述了主要涉及分析软骨特异性基质蛋白及其相应mRNA合成的表型评估技术。还描述了研究基因调控的方法，包括用转录和信号调节因子的表达载体转染启动子驱动的报告基因、染色质免疫沉淀和DNA甲基化。
