[应用实时定量PCR技术对类风湿性关节炎滑膜细胞中诱导肿瘤坏死因子-α及白细胞介素-1β的白三烯B4 mRNA表达水平进行定量分析]

[Quantification of expression of leukotriene B4 inducing tumor necrosis factor-alpha and interleukin-1beta at mRNA level in synovial membrane cells of rheumatoid arthritis by real-time quantitative PCR].

作者信息

Chen Zhan-kun, Lv Hou-shan

机构信息

Arthritis Recearch Center, Peking University Peopleos Hospital, Beijing 100044,China.

出版信息

Beijing Da Xue Xue Bao Yi Xue Ban. 2006 Oct 18;38(5):533-6.

PMID:17068631

Abstract

OBJECTIVE

To investigate quantification of expression of LTB4 inducing IL-1beta and TNF-alpha 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-alpha and IL-1beta were detected at mRNA level by Real-time Quantitative PCR.

RESULTS

Expressions of basic TNF-alpha (TNF-alpha/GAPDH) and IL-beta (IL-beta/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) mol/L) was shown to induce dose-dependent increase of mRNA expression of TNF-alpha. (7-15 times) and IL-1beta (1 time) , endogenous product of LTB4 by LIT significantly increased mRNA expressions of TNF-alpha (145 times) and IL-1beta (12 times) respectively. LIT-treated synoviocytes with addition of MK-886 (5-LOX exciting protein FLAP inhibitor) (1-10 micromol/L) were inhibited to secrete LTB4 dose-dependently, following the markedly down-regulated expressions of TNF-alpha (15%-66%) and IL-1beta (41%-71%) at mRNA level . Bestatin(100 mg/L) could also remarkably diminish LTB4-induced mRNA expressions of TNF-alpha(86%) and IL-1beta (79%).

CONCLUSION

LTB4 of synovial membrance cells in rheumatoid arthritis could induce expressions of TNF-alpha and IL-1beta 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.

摘要

目的

研究类风湿关节炎滑膜细胞中白三烯B4（LTB4）诱导白细胞介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）在mRNA水平表达的定量情况。

方法

分别在白细胞介素-1（LIT）存在的情况下，用外源性LTB4、MK-886（5-脂氧合酶激活蛋白抑制剂）和贝司他汀（白三烯A4水解酶抑制剂）处理类风湿关节炎患者原代培养的滑膜细胞，通过实时定量聚合酶链反应（Real-time Quantitative PCR）在mRNA水平检测TNF-α和IL-1β的表达。

结果

原代培养滑膜细胞中基础TNF-α（TNF-α/甘油醛-3-磷酸脱氢酶（GAPDH））和IL-1β（IL-1β/GAPDH）在mRNA水平的表达分别为0.02±0.00和0.16±0.01。LTB4（10⁻⁹mol/L - 10⁻⁸mol/L）呈剂量依赖性诱导TNF-α mRNA表达增加（7 - 15倍）和IL-1β mRNA表达增加（1倍），LIT产生的内源性LTB4产物分别显著增加TNF-α mRNA表达（145倍）和IL-1β mRNA表达（12倍）。添加MK-886（5-脂氧合酶激动蛋白FLAP抑制剂）（1 - ～10μmol/L）处理LIT刺激的滑膜细胞，可剂量依赖性抑制LTB4分泌，随后TNF-α mRNA表达显著下调（15% - 66%），IL-1β mRNA表达显著下调（41% - 71%）。贝司他汀（100mg/L）也可显著降低LTB4诱导的TNF-α mRNA表达（86%）和IL-1β mRNA表达（79%）。