Supplementary Material for: Phenotypic Characterization of Human Chondrocyte Cell Line C-20/A4: A Comparison between Monolayer and Alginate Suspension Culture

DNA microarray analysis was used to investigate the molecular phenotype of one of the first human chondrocyte cell lines, C-20/A4, derived from juvenile costal chondrocytes by immortalization with origin-defective simian virus 40 large T antigen. Clontech Human Cancer Arrays 1.2 and quantitative PCR were used to examine gene expression profiles of C-20/A4 cells cultured in the presence of serum in monolayer and alginate beads. In monolayer cultures, genes involved in cell proliferation were strongly upregulated compared to those expressed by human adult articular chondrocytes in primary culture. Of the cell cycle-regulated genes, only two, the CDK regulatory subunit and histone H4, were downregulated after culture in alginate beads, consistent with the ability of these cells to proliferate in suspension culture. In contrast, the expression of several genes that are involved in pericellular matrix formation, including MMP-14, COL6A1, fibronectin, biglycan and decorin, was upregulated when the C-20/A4 cells were transferred to suspension culture in alginate. Also, nexin-1, vimentin, and IGFBP-3, which are known to be expressed by primary chondrocytes, were differentially expressed in our study. Consistent with the proliferative phenotype of this cell line, few genes involved in matrix synthesis and turnover were highly expressed in the presence of serum. These results indicate that immortalized chondrocyte cell lines, rather than substituting for primary chondrocytes, may serve as models for extending findings on chondrocyte function not achievable by the use of primary chondrocytes.

本研究采用DNA微阵列分析（DNA microarray），探究首批人软骨细胞系之一C-20/A4的分子表型；该细胞系通过缺陷型猿猴病毒40大T抗原（origin-defective simian virus 40 large T antigen）永生化，源自幼年肋软骨细胞。研究使用Clontech人类癌症基因芯片1.2版（Human Cancer Arrays 1.2）与定量聚合酶链式反应（quantitative PCR），检测在血清存在条件下于单层培养及海藻酸微珠中培养的C-20/A4细胞的基因表达谱。在单层培养体系中，与原代培养的成人关节软骨细胞相比，参与细胞增殖的基因呈现显著上调。在细胞周期调控基因中，仅细胞周期蛋白依赖性激酶调控亚基与组蛋白H4（histone H4）在海藻酸微珠中培养后出现下调，这与该细胞在悬浮培养中仍可增殖的特性相符。与之相反，当C-20/A4细胞被转移至海藻酸微珠悬浮培养体系时，多种参与细胞周围基质形成的基因表达上调，包括基质金属蛋白酶14（MMP-14）、Ⅵ型胶原蛋白α1链（COL6A1）、纤连蛋白、饰胶蛋白聚糖与核心蛋白聚糖。此外，已知由原代软骨细胞表达的连接蛋白-1（nexin-1）、波形蛋白（vimentin）与胰岛素样生长因子结合蛋白3（IGFBP-3）在本研究中呈现差异表达。与该细胞系的增殖表型一致，在血清存在条件下，参与基质合成与代谢的基因鲜有高表达。本研究结果表明，永生化软骨细胞系虽无法替代原代软骨细胞，但可作为模型，用于拓展原代软骨细胞研究难以实现的软骨细胞功能相关发现。