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Gene expression profiling of IL-1β-stimulated synovial cells from TMJ using Gene Chip

使用基因芯片对 IL-1β 刺激的 TMJ 滑膜细胞进行基因表达谱分析

基本信息

批准号：
14571915
负责人：
OGURA Naomi
金额：
$ 2.24万
依托单位：
Nihon University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

Interleukin-1β(IL-1β) has important roles in the inflammation and connective tissue destruction observed in joint diseases such as rheumatoid arthritis. IL-1β is also a key mediator of intracapsular pathologic conditions of the temporomandibular joint(TMJ), including disk displacement/internal derangement and osteoarthritis. To identify putative IL-1β-responsive genes from arthritic disease tissues, we investigated the effects of IL-1β on gene expression of 8,793 genes in synovial fibroblasts from five TMJ patients. <Methods>Synovial fibroblasts were obtained using an outgrowth method with synovial tissue from a patient who underwent arthrotomy of the TMJ. Total RNA of synovial fibroblasts, incubated with or without i□unit/ml of IL-1β for 4 hours, was extracted using the RNeasy kit. Gene expression pro filing was used GeneChip (HG Focus Array,8,763 genes).Hybridization data were analyzed using Gene Springs^<TM> software. Gene expression levels were also confirmed by real time-PCR. <results>Significant changes between control and IL-1β-treated cells (p<0.05) were detected in 170 genes:139 up-regulated genes and 31 down-regulated genes. The most enhanced gene by IL-1β was CCL20, which is one of chemokines. The 170 IL-1β-responsive genes included 9 chemokines. Real-time PCR confirms that mRNA levels of these chemokines increase after IL-1β treatment. In addition, the effects of IL-1β were investigated using Ingenuity Pathway Analysis. We found that IL-1β affects the expression of several genes in the NFκB signaling pathway and were able to confirm that mRNA levels of NFκB1 and IκBα increase after IL-1β treatment by endpoint-PCR and real time-PCR analysis. The results suggest that IL-1β-responsive genes play important roles in progression of inflammation and destruction of joint components.

白细胞介素-1 β（IL-1β）在类风湿性关节炎等关节疾病中观察到的炎症和结缔组织破坏中具有重要作用。IL-1β也是颞下颌关节（TMJ）囊内病理状况的关键介质，包括盘移位/内部紊乱和骨关节炎。为了从关节炎疾病组织中鉴定假定的IL-1β应答基因，我们研究了IL-1β对5名TMJ患者滑膜成纤维细胞中8，793个基因表达的影响。<Methods>滑膜成纤维细胞获得使用的方法与滑膜组织从谁经历了关节切开术的颞下颌关节的患者。使用RNeasy试剂盒提取滑膜成纤维细胞的总RNA，用或不用1 □单位/ml的IL-1β孵育4小时。采用基因芯片（HG Focus Array，8，763个基因）进行基因表达谱分析<TM>。基因表达水平也通过真实的时间-PCR证实。<results>在对照和IL-1β处理的细胞之间，在170个基因中检测到显著变化（p<0.05）：139个上调基因和31个下调基因。IL-1β对趋化因子CCL 20表达的增强作用最强。170个IL-1β应答基因中包括9个趋化因子。实时PCR证实IL-1β处理后这些趋化因子的mRNA水平增加。此外，使用损伤途径分析来研究IL-1β的作用。我们发现IL-1β影响NFκB信号通路中几个基因的表达，并且能够通过终点PCR和真实的时间PCR分析证实IL-1β处理后NFκB1和IκBα的mRNA水平增加。结果表明，IL-1β反应基因在炎症进展和关节组件破坏中起重要作用。