Robbins J R, Thomas B, Tan L, Choy B, Arbiser J L, Berenbaum F, Goldring M B
Beth Israel Deaconess Medical Center, and New England Baptist Bone & Joint Institute, Boston, Massachusetts, USA.
Arthritis Rheum. 2000 Oct;43(10):2189-201. doi: 10.1002/1529-0131(200010)43:10<2189::AID-ANR6>3.0.CO;2-S.
To develop a reproducible immortalized human chondrocyte culture model for studying the regulation of chondrocyte functions relevant to arthritic diseases in adult humans.
Primary adult articular chondrocytes were immortalized with a retrovirus expressing a temperature-sensitive mutant of SV40-large T antigen (tsTAg). The established tsT/AC62 chondrocyte cell line was examined in monolayer and alginate culture systems. The levels of messenger RNA (mRNA) encoding cartilage matrix proteins and interleukin-1beta (IL-1beta)-inducible mRNA were analyzed by reverse transcriptase-polymerase chain reaction. Matrix protein synthesis was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 35S-sulfate-labeled proteoglycans and Western blotting of type II collagen and aggrecan. Type II collagen (COL2A1)-luciferase reporter gene expression was analyzed by transient transfection. Phosphorylated stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38 mitogen-activated protein kinase (p38 MAPK), and activating transcription factor 2 (ATF-2) were detected by Western blotting.
The tsT/AC62 cells expressed TAg at the permissive temperature (32degrees C), and the loss of TAg at 37 degrees C and 39 degrees C correlated with decreased cell proliferation. Cells in alginate culture deposited abundant alcian blue-stainable matrix and continued to proliferate at 32 degrees C. Preferential retention of aggrecan was observed in the cell-associated matrix, while biglycan and decorin were secreted into the medium of monolayer and alginate cultures. The levels of COL2A1 and aggrecan mRNA were increased after transfer from monolayer to alginate culture at 32 degrees C. Treatment with IL-1beta decreased COL2A1 and aggrecan mRNA levels and increased the levels of matrix metalloproteinases 1, 3, and 13 mRNA, as well as those of cyclooxygenase 2, type I collagen, and secretory phospholipase A2 type IIA mRNA, but not those of inducible nitric oxide synthase mRNA. IL-1beta also stimulated phosphorylation of p38 MAPK, SAPK/JNK, and ATF-2. The p38 MAPK-selective inhibitor, SB203580, partially reversed IL-1beta-induced inhibition of COL2A1 mRNA levels and COL2A1-luciferase reporter gene expression.
The tsT/AC62 cells provide a reproducible model that mimics the adult articular chondrocyte phenotype, particularly in alginate culture, and demonstrates characteristic responses to IL-1beta. These studies also show, for the first time, that p38 MAPK is one of the signals required for IL-1beta-induced inhibition of COL2A1 gene expression. Availability of this model will permit identification of signals that regulate cytokine responses, and will also provide rational strategies for targeting these pathways.
建立一种可重复的永生化人软骨细胞培养模型，用于研究与成年人类关节炎疾病相关的软骨细胞功能调控。
用表达SV40大T抗原温度敏感突变体（tsTAg）的逆转录病毒使原代成年关节软骨细胞永生化。在单层培养和藻酸盐培养系统中对建立的tsT/AC62软骨细胞系进行检测。通过逆转录聚合酶链反应分析编码软骨基质蛋白的信使核糖核酸（mRNA）水平以及白细胞介素-1β（IL-1β）诱导的mRNA水平。通过对35S-硫酸盐标记的蛋白聚糖进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳以及对II型胶原蛋白和聚集蛋白聚糖进行蛋白质印迹分析基质蛋白合成。通过瞬时转染分析II型胶原蛋白（COL2A1）-荧光素酶报告基因表达。通过蛋白质印迹检测磷酸化应激激活蛋白激酶/c-Jun氨基末端激酶（SAPK/JNK）、p38丝裂原活化蛋白激酶（p38 MAPK）和活化转录因子2（ATF-2）。
tsT/AC62细胞在允许温度（32℃）下表达TAg，在37℃和39℃时TAg的缺失与细胞增殖减少相关。藻酸盐培养中的细胞沉积了丰富的阿尔辛蓝可染色基质，并在32℃下继续增殖。在细胞相关基质中观察到聚集蛋白聚糖的优先保留，而双糖链蛋白聚糖和核心蛋白聚糖分泌到单层培养和藻酸盐培养的培养基中。在32℃从单层培养转移到藻酸盐培养后，COL2A1和聚集蛋白聚糖mRNA水平升高。用IL-1β处理降低了COL2A1和聚集蛋白聚糖mRNA水平，并增加了基质金属蛋白酶1、3和13 mRNA水平，以及环氧合酶2、I型胶原蛋白和分泌型磷脂酶A2 IIA型mRNA水平，但不影响诱导型一氧化氮合酶mRNA水平。IL-1β还刺激了p38 MAPK、SAPK/JNK和ATF-2的磷酸化。p38 MAPK选择性抑制剂SB203580部分逆转了IL-1β诱导的COL2A1 mRNA水平抑制和COL2A1-荧光素酶报告基因表达。
tsT/AC62细胞提供了一个可重复的模型，该模型模拟成年关节软骨细胞表型，特别是在藻酸盐培养中，并显示出对IL-1β的特征性反应。这些研究还首次表明，p38 MAPK是IL-1β诱导的COL2A1基因表达抑制所需的信号之一。该模型的可用性将允许识别调节细胞因子反应的信号，并将为靶向这些途径提供合理策略。
