[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].
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%).


CONCLUSIONS
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.
Chen
Zhan-kun
ZK
Arthritis Recearch Center, Peking University Peopleos Hospital, Beijing 100044,China.
Lv
Hou-shan
HS
D001172:Arthritis, Rheumatoid / Q000235:genetics / Q000473:pathology; D002478:Cells, Cultured; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D053583:Interleukin-1beta / Q000235:genetics*; D007975:Leukotriene B4 / Q000494:pharmacology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D013583:Synovial Membrane / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*; D002722:Chlorobenzenes
D007211:Indoles; D053583:Interleukin-1beta; D007975:Leukotriene B4; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C060893:MK-886; D002722:Chlorobenzenes