类风湿关节炎滑膜细胞中白三烯B4诱导肿瘤坏死因子-α和白细胞介素-1β mRNA表达的测定  

Quantification of expression of leukotriene B4 inducing tumor necrosis factor-α and interleukin-1β at mRNA level in synovial membrane cells of rheumatoid arthritis by real-time quantitative PCR

作　　者：陈占昆[1] 吕厚山[1] 

机构地区：[1]北京大学人民医院关节病研究中心,北京100044

出　　处：《北京大学学报（医学版）》2006年第5期533-536,共4页Journal of Peking University:Health Sciences

基　　金：国家重点基础研究发展规划项目(2002CB513007)资助~~

摘　　要：目的:探讨类风湿关节炎(rheumatoid arthritis,RA)滑膜细胞中白三烯B4(LTB4)诱导肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)mRNA表达的定量测定方法。方法:RA患者的滑膜细胞进行原代培养,加入外源性LTB4或者在LIT存在的情况下,分别加入MK-886(5-脂氧合酶激动蛋白抑制剂)和苯丁抑制素(Bestatin,LTA4水解酶抑制剂),采用TaqMan PCR来定量检测滑膜细胞中TNF-α和IL-1βmRNA水平的表达。结果:原代培养的RA滑膜细胞的基本TNF-α和IL-1βmRNA表达水平(TNF-α/GAPDH和IL-1β/GAPDH),分别为0.02±0.00和0.16±0.01。当加入外源性的LTB410-9和10-8mol/L后,LTB410-9mol/L使TNF-αRNA水平的表达增高了7倍,LTB410-8mol/L使TNF-αmRNA水平的表达增高了15倍,LTB410-8mol/L使IL-1βmRNA水平增高了1倍。而加入LIT刺激内源性的LTB4增高后,使TNF-α和IL-1βmRNA水平分别增高了145倍和12倍。在LIT存在的情况下,加入LTB4合成抑制剂MK-886(1μmol/L,10μmol/L)后,使TNF-αmRNA水平的表达分别下降15%和66%,IL-1βm RNA水平的表达分别下降了41%和71%。1100mg/L苯丁抑制剂使TNF-α和IL-1βmRNA水平表达分别降低了86%和79%。结论:RA滑膜细胞中LTB4可诱导TNF-α和IL-1βmRNA水平的表达,并有助于定量测定mRNA表达水平。Objective： To investigate quantification of expression of LTB4 inducing IL-1β and TNF-α at mRNA level in synovial membrane cells of rheumatoid arthritis. Methods： Primary cultured synovial cells from RA patients were treated with exogenous LTB4, MK-886 （inhibitor of 5-lipoxygenase activating protein） and Bestatin （ inhibitor of leukotriene A4 hydrolase） in the presence of LIT respectively, expressions of TNF-α and IL-1β were detected at mRNA level by Real-time Quantitative PCR. Results： Expressions of basic TNF-α （TNF-α/GAPDH） and IL-β （IL-β/GAPDH） at mRNA level in primary cultured synovial cells were 0.02±0.00 and 0.16 ±0.01 respectively. LTB4（ 10^-9 mol/L - 10^-8 tool/L） was shown to induce dose-dependent increase of mRNA expression of TNF-α. （7 -15 times） and IL-1β （1 time） , endogenous product of LTB4 by LIT significantly increased mRNA expressions of TNF-α （145 times） and IL-1β （12 times） respectively. LIT-treated synoviocytes with addition of MK-886 （5-LOX exciting protein FLAP inhibitor） （ 1 - 10 μmol/L） were inhibited to secrete LTB4 dose-dependently, following the markedly down-regulated expressions of TNF-α （15% -66% ） and IL-1β （41% - 71% ） at mRNA level . Bestatin（100 mg/L） could also remarkably diminish LTB4-induced mRNA expressions of TNF-α（86% ） and IL-1β （79%）. Conclusion： LTB4 of synovial membrance cells in rheumatoid arthritis could induce expressions of TNF-α and IL-1β at mRNA level, and their expression at mRNA level had been quantified successfully. It is a beneficial help to quantify all kinds of cytokines in methodology.

关 键 词：白三烯B4 肿瘤坏死因子Α 白细胞介素1 关节炎 类风湿 聚合酶链反应 

分 类 号：R593.22[医药卫生—内科学]