摘要
目的通过体外培养骨关节炎（OA）软骨细胞体系，了解肿瘤坏死因子（TNF）-α和白细胞介素（IL）-1β对蛋白聚糖代谢的调节作用。方法软骨细胞来自OA患者进行膝关节置换术的股骨髁．实验采用第1代软骨细胞。随机将细胞分为4组：对照组、TNF-α组、IL-1β组和联合组（TNF—α＋IL-1β），在培养液中加入不同的刺激因子共培养4d。采用3种方法了解蛋白聚糖的代谢水平：①用二甲基亚甲蓝分光光度法（DMMB法）检测细胞中及培养液中的糖胺多糖（GAG）含量；②酶联免疫吸附法通过2种抗蛋白聚糖单克隆抗体（Mab-383和5D4）检测培养液中蛋白聚糖的不同代谢片段；③反转录-聚合酶链式反应（RT—PCR）半定量法检测软骨细胞的蛋白聚糖、带有血小板凝血酶敏感蛋白样模体的解整链蛋白金属蛋白酶-4（ADAMTS-4）和ADAMTS-5的mRNA水平。结果①实验组培养液中GAG含量均明显高于对照组（P〈0．01）；②TNF-α组、IL-1β组及联合组的培养液中5D4片段的水平均高于对照组（P〈0．01），而各组383片段水平差异无统计学意义；③实验组的蛋白聚糖mRNA水平均低于对照组（P〈0．01），TNF—α组的ADAMTS-5、IL-1β组的ADAMTS-4及联合组的两种酶的mRNA水平均显著高于对照组（P〈0．01）。结论TNF-α及IL-1β可下调入OA软骨细胞的蛋白聚糖mRNA表达，上调ADAMTS-4及ADAMTS-5的mRNA表达，并促进蛋白聚糖的分解，可能在OA的发病中起着一定的作用。
Objective To evaluate the effects of tumor necrosis factor （TNF）-alpha and interleukin （IL）-1 beta on aggrecan metabolism in cultured human osteoarthritis （OA） chondrocytes. Methods Chondrocytes were cultured with or without TNF-α and/or IL-1 for four days. Glycosaminoglycan （GAG） content in the chondrocytes and matrix were measured by 1,9-dimethylmethylene blue spectrophotometric assay. Aggrecan catabolic fragments in matrix were measured by ELISA using anti-aggrecan monoclonal antibodies 3B3 and 5D4. RNA was isolated from chondrocytes and analyzed for the expression of aggrecan, disintegrin A and metalloproteinase with thrombospondin motifs-4 （ADAMTS-4）, ADAMTS-5 and 18S as control by reverse transcription-polymerase chain reaction （RT-PCR）. Results Levels of GAG in matrix samples of the three groups （TNF-α, IL-1beta and the combination of these two） increased significantly comparing with the control group （P〈0.01）. Similiar increase could be observed in fragments of 5D4 in matrix of the three groups but there was no difference could be observed in the 3B3 fragments among the four groups. Levels of aggrecan mRNA in the three groups were lower than those of the control （P〈0.01）. mRNA of ADAMTS-4 of the IL-1 beta group and mRNA of ADAMTS-5 of the TNF-α group were higher than those of the control group （P〈0.01）. Conclusion The results suggest that TNF-α and IL-1beta can stimulate aggrecan catabolism. ADAMTS-4 and ADAMTS-5 may play a role in the pathogenesis of OA and may represent a potential target for the treatment of OA.
出处
《中华风湿病学杂志》
CAS
CSCD
2008年第2期83-87,共5页
Chinese Journal of Rheumatology
