ABSTRACT
Objective Establishment of two experimental models for osteoclast differentiation from monocyte in vitro,and to study the potential of osteoclast differentiation induced by cytokines.Methods Direct model of osteoclast differentiation: CD14+ monocyte fraction of peripheral blood mononuclear cell(PBMC) stimulated by(25 ?g/L) M-CSF+(10~(-8)mol/L) LTB4 for two weeks.Indirect model of osteoclast differentiation: Utilize the coculture model of RAFLs and monocyte that were stimulated in the presence of 25 g/L M-CSF+(10~(-8)mol/L) LTB4 for three weeks.In TRAP staining the multinucleated TRAP staining positive osteoclast-like cells were counted as marker of as differentiation effect of each group.Results Osteoclast-like cells can be induced by both direct and indirect models.Conclusion Two experimental models for osteoclast differentiation can be separately used to study the effect of various cytokines for direct and indirect OC differentiation.
ABSTRACT
Objective:To explore the effect of progrmmed cell death 5(PDCD5)on apoptosis of rheumatoid arthritis fibroblast-like synoviocytes(RA FLS) induced by triptolide.Methods:Cultured synovial cells in vitro from RA patients were transfected with Ad-PDCD5.At protein level,expression of PDCD5 in RA FLS infected with Ad-PDCD5 was detected by Western blot.RA FLS infected with Ad-PDCD5 were cultured in presence or absence of triptolide and apoptosis of RA FLS was determined by flow cytometry.Results:Infection of RA FLS with increasing concentrations of Ad-PDCD5(50-300 MOI) resulted in a does-dependent increase in the production of PDCD5.Apoptotic cells percentage for noinfection group,Ad-null group and Ad-PDCD5 group were(22.41?3.87)%,(28.77?12.97)% and(48.87?12.69)%,respectively.Alternatively,infection without addition of triptolide stimuli had no effect.The data showed that gene transfection of PDCD5 alone without addition of triptolide was not sufficient to activate RA FLS apoptosis,PDCD5 acted as an enhancer rather than inductor of apoptosis.Conclusion:Overexpression of PDCD5 could enhance apoptosis of RA FLS induced by triptolide,PDCD5 may be a potential therapeutic target to RA.
ABSTRACT
Objective: To investigate quantification of expression of LTB4 inducing IL-1? and TNF-? 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-? and IL-1? were detected at mRNA level by Real-time Quantitative PCR. Results: Expressions of basic TNF-?(TNF-?/GAPDH) and IL-?(IL-?/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-?.(7-15 times) and IL-1? (1 time) , endogenous product of LTB4 by LIT significantly increased mRNA expressions of TNF-? (145 times) and IL-1?(12 times) respectively. LIT-treated synoviocytes with addition of MK-886 (5-LOX exciting protein FLAP inhibitor) (1-10 ?mol/L) were inhibited to secrete LTB4 dose-dependently, following the markedly down-regulated expressions of TNF-? (15%-66%) and IL-1? (41%-71%) at mRNA level . Bestatin(100 mg/L) could also remarkably diminish LTB4-induced mRNA expressions of TNF-?(86%) and IL-1?(79%). Conclusion: LTB4 of synovial membrance cells in rheumatoid arthritis could induce expressions of TNF-? and IL-1? 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.
ABSTRACT
<p><b>OBJECTIVE</b>To confirm the role of HLA-B2704 and hbeta(2)m gene in the pathogenesis of spontaneous inflammatory diseases by establishing HLA-B2704 and hbeta(2)m double transgenic mice model of ankylosing spondylitis. It will provide a powerful animal model for exploring the etiology, prevention and treatment of B27-relevant diseases.</p><p><b>METHODS</b>The screening, identification and expression of HLA-B2704 and hbeta(2)m gene were determined by PCR, dot blot, Southern blot hybridization, RT-PCR, flow cytometry and immunohistochemistry. HE staining was performed for the diseased mice.</p><p><b>RESULTS</b>Eight double transgenic mice bearing high copy developed spontaneous dermatosis, arthritis and nail changes in the rear paw. The results of flow cytometry in normal mice, B27 single transgenic mice, and HLA-B27/hbeta(2)m double transgenic mice were 0.63%, 7.87% and 35.87% respectively. HLA-B2704 antigen was high expressed on the cell surface, but not evident on those of B27 single transgenic mice.</p><p><b>CONCLUSIONS</b>HLA-B2704 heavy chain can induce spontaneous inflammatory diseases in the transgenic mice. Hbeta(2)m can form a stable complex with HLA-B27 and may stabilize and enhance the expression of HLA-B2704 on the cell surface.</p>
Subject(s)
Animals , Mice , Disease Models, Animal , HLA-B27 Antigen , Genetics , Inflammation , Mice, Transgenic , Reverse Transcriptase Polymerase Chain Reaction , Spondylitis, Ankylosing , GeneticsABSTRACT
Objective To investigate the expression of extracellular matrix metalloproteinase induced (EMMPRIN) in the synovial membrane of patients with rheumatoid arthritis (RA).Methods Immunohistochemistry were performed to characterize the EMMPRIN-expressing cells at sites of joint destruction derived from RA and osteoarthritis (OA) patients.Reverse trancription polymerase chain reaction (RT-PCR) was performed to study the existence of EMMPRIN in synovial membrane samples.And it was followed by computer assisted image analysis in order to detect the A values of their experession.Results EMMPRIN immunoreactivity was more intense in RA than in OA synovial membrane (P