Jeong Jae-Gyun, Kim Jong-Mook, Cho Hongchan, Hahn Woong, Yu Seung Shin, Kim Sunyoung
Institute of Molecular Biology and Genetics, School of Biological Sciences, Seoul National University, Seoul 151-742, Republic of Korea.
Biochem Biophys Res Commun. 2004 Nov 5;324(1):3-7. doi: 10.1016/j.bbrc.2004.09.011.
IL-1 is one of the key mediators involved in the pathogenesis of rheumatoid arthritis (RA) and is known to affect the level of gene expression in various settings. We investigated the effects of IL-1beta on the expression of 240 genes in rheumatoid synovial fibroblasts (RSFs) using a cDNA microarray. Total RNAs were prepared from RSFs stimulated with IL-1beta and hybridized to the microarray. The fluorescence intensity of each gene was compared between the control and IL-1beta-treated cells. To confirm the data obtained from the microarray analysis, the level of gene expression was also examined by ELISA, Northern blot, or Western blot depending on the genes to be analyzed. The genes whose levels were significantly changed by IL-1beta in the microarray analysis could be divided into three categories; inflammatory mediators, matrix-modifying enzymes, and apoptosis-associated molecules. The increase in the mRNA levels of IL-6, IL-8, MCP-1, and GRO-1 was confirmed by determining their protein levels from the cell culture supernatant using ELISA. The increase in the level of two matrix-degrading enzymes, MMP-1 and MMP-3, was reproducibly observed by an ELISA method, while the decrease in the level of TIMP-3, an inhibitor of MMPs, was confirmed by Northern blot analysis. The fluorescence intensity of two apoptosis-related genes, caspase-3 and Bcl-xL, was significantly lowered. The decreased protein level of caspase-3 was also found. Our data suggested that IL-1beta could provoke a series of responses in RSFs leading to the pathologic status of RA, including enhancement of inflammatory cytokines, imbalanced production of MMPs and TIMPs, and dysregulation of apoptosis.
白细胞介素 -1（IL -1）是类风湿性关节炎（RA）发病机制中涉及的关键介质之一，已知其在多种情况下会影响基因表达水平。我们使用cDNA微阵列研究了IL -1β对类风湿性滑膜成纤维细胞（RSF）中240个基因表达的影响。从用IL -1β刺激的RSF中制备总RNA，并与微阵列杂交。比较了对照细胞和经IL -1β处理的细胞中每个基因的荧光强度。为了确认从微阵列分析获得的数据，还根据要分析的基因通过酶联免疫吸附测定（ELISA）、Northern印迹或Western印迹检查基因表达水平。在微阵列分析中其水平因IL -1β而发生显著变化的基因可分为三类：炎症介质、基质修饰酶和凋亡相关分子。通过使用ELISA从细胞培养上清液中测定其蛋白质水平，证实了IL -6、IL -8、单核细胞趋化蛋白 -1（MCP -1）和生长调节致癌基因 -1（GRO -�1）的mRNA水平升高。通过ELISA方法可重复性地观察到两种基质降解酶基质金属蛋白酶 -1（MMP -1）和基质金属蛋白酶 -3（MMP -3）水平的升高，而通过Northern印迹分析证实了基质金属蛋白酶组织抑制剂 -3（TIMP -3）水平的降低，TIMP -3是MMPs的一种抑制剂。两种凋亡相关基因半胱天冬酶 -3（caspase -3）和Bcl -xL的荧光强度显著降低。还发现了caspase -3蛋白质水平的下降。我们的数据表明，IL -1β可在RSF中引发一系列反应，导致RA的病理状态，包括炎症细胞因子的增强、MMPs和TIMPs的产生失衡以及细胞凋亡的失调。
