McDougall S, Fu Y H, Lowe G N, Williams A, Polendo R, Benya P D, Iida-Klein A, Fang M A, Hahn T J
Department of Medicine, West Los Angeles VA Medical Center, California, USA.
J Bone Miner Res. 1996 Aug;11(8):1130-8. doi: 10.1002/jbmr.5650110812.
Recent evidence suggests that decreased chondrocyte function in osteoarthritis and other articular disorders may be due to chondrocyte dedifferentiation produced by altered regulatory signals from the cartilage extracellular matrix (ECM). However, there are currently no mammalian chondrocytic cell line systems adapted to the study of this process. We therefore examined the effects of ECM growth conditions on markers of differentiated chondrocytic phenotype expression in the nontransformed rat RCJ 3.1C5.18 (RCJ) chondrocyte cell line, including type II collagen expression, aggrecan production, link protein gene expression, and parathyroid hormone (PTH) receptor number. RCJ cells grown in monolayer on plastic exhibited a dedifferentiated phenotype characterized by flattened cell morphology, with > 80% type I collagen and < 5% type II collagen production, as determined by two-dimensional gel mapping electrophoresis of collagen cyanogen bromide peptides. In addition, aggrecan production was low, and link protein mRNA was not expressed at detectable levels. After transfer to growth under minimal attachment conditions on the surface of a composite type I collagen/agarose (0.15%-0.8%) gel (CAG) for 7 days, RCJ cells developed a rounded, chondrocytic morphology and a pattern of differentiated, chondrocytic gene expression, with 79% type II and 8% type I collagen production. Steady-state type I and type II procollagen mRNA levels were altered in parallel with collagen protein expression. In cells grown on CAG, aggrecan production increased 6-fold, and there was a marked increase in both aggrecan core protein and link protein mRNA levels. In addition, maximal PTH-stimulated cAMP generation increased 15-fold in association with an increased PTH receptor number. Therefore, the RCJ chondrocyte cell line is highly sensitive to ECM regulation of chondrocyte-specific gene expression.
近期证据表明，骨关节炎及其他关节疾病中软骨细胞功能的下降可能是由于软骨细胞去分化所致，而这种去分化是由软骨细胞外基质（ECM）调控信号改变所引起的。然而，目前尚无适用于研究此过程的哺乳动物软骨细胞系系统。因此，我们研究了ECM生长条件对未转化的大鼠RCJ 3.1C5.18（RCJ）软骨细胞系中分化软骨细胞表型表达标志物的影响，这些标志物包括II型胶原蛋白表达、聚集蛋白聚糖产生、连接蛋白基因表达以及甲状旁腺激素（PTH）受体数量。在塑料上单层培养的RCJ细胞呈现去分化表型，其特征为细胞形态扁平，通过溴化氰胶原蛋白肽的二维凝胶图谱电泳测定，I型胶原蛋白产生量>80%，II型胶原蛋白产生量<5%。此外，聚集蛋白聚糖产生量低，连接蛋白mRNA未检测到表达。在I型胶原蛋白/琼脂糖（0.15%-0.8%）复合凝胶（CAG）表面以最小附着条件培养7天后，RCJ细胞呈现圆形软骨细胞形态以及分化的软骨细胞基因表达模式，II型胶原蛋白产生量为79%，I型胶原蛋白产生量为8%。I型和II型前胶原蛋白mRNA的稳态水平与胶原蛋白蛋白表达平行改变。在CAG上培养的细胞中，聚集蛋白聚糖产生量增加了6倍，聚集蛋白聚糖核心蛋白和连接蛋白mRNA水平均显著增加。此外，最大PTH刺激的cAMP生成增加了15倍，同时PTH受体数量增加。因此，RCJ软骨细胞系对ECM对软骨细胞特异性基因表达的调控高度敏感。
