The Establish Of The Human Fibroblast-like Synoviocytes Culture With Rheumatoid Arthritis In Itro And The Deternination Of The Expression Of RANKL In Them By Stimulation Of Interleukin-23p19

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by progressively chronic joint inflammation, synovial hyperplasia, and destruction of articular cartilage and bone, and finally disfiguration and functional disability of the majority of joints. Recent researches had found that fibroblast-like synoviocytes (FLS) played important role in the establishment and maintenance of RA, and they were the basic elements for RA pathogenesis.IL-23is a heterodimeric pro-inflammatory cytokine belonging to the IL-12family, secreted by activated dendritic cells (DCs) and macrophages, and it is an essential factor for the maintenance and survival of Th17cells. The levels of IL-23in the synovial fluid, serum, and synovial tissue of RA patients were strongly associated with disease activity in RA. A recent study showed that IL-23blockade by IL-23receptor results to obvious decreases in the production of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6.Receptor activator of nuclear factor (NF) κB ligand (RANKL) is a key molecule for osteoclast differention, which is expressed locally in the synovial tissue of RA patients. FLS express RANKL and can thus drive osteoclast formation. This study was undertaken to explore the role of IL-23in stimulating the expression of RANKL and to study the mechanism in this process. Part Ⅰ. The establishment of the human fibroblast-like synoviocytes culture with rheumatoid arthritis in vitroObjective:To establish the human fibroblast-like synoviocytes culture with rheumatoid arthritis in vitro.Methods:The synovial tissues were obtained from13rheumatoid arthrits (RA) patients, and FLS were isolated in vitro. FLS were detected by cell morphology, flow cytometry and western blot.Results:The FLS were successfully cultured from synovial membrance tissues in vitro. Flow cytometry showed that FLS from RA patients contained96.3%CD90cells. The protein of CD90was expressed in FLS.Conclusion:FLS from RA patients can be effectively and easily cultured by means of tissue culture.Part Ⅱ. The expression of RANKL in FLS was upregulated by stimulation of interleukin-23p19Objective:To explore the expression of RANKL in FLS by stimulation of interleukin-23Methods:FLS were stimulated with various concentrations IL-23p19(0,1,5,20ng/ml). After incubating for24hours, the level of RANKL mRNA was measured by real time PCR. Then, FLS were stimulated with IL-23p19(5ng/ml) after the incubation for (0,1,4,8,12,24h), and RANKL mRNA was measured by real time-PCR and protein was detected by western blot. FLS were stimulated with5ng/mlIL-23p19,1ng/ml IL-17A,2ng/mlIL-17F,5ng/ml IL-23p19jointed withlng/ml IL-17A and5ng/ml IL-23p19jointed with2ng/ml IL-17F; the level of RANKL was detected by real time-PCR. Then IL-17R, IL-23R, IL-12Rβ1and IL-12Rβ2mRNA expression was detected by real time-PCR after FLS were stimulated with5ng/mlIL-23p19,1ng/ml IL-17A and2ng/mlIL-17F. Then FLS were stimulated with inhibitor of JNK, inhibitor of Erk, inhibitor of JAK2, inhibitor of JAK3, inhibitor of NF-κB, inhibitor of PI3K, after incubating with inhibitors for1hour, and FLS were stimulated with IL-23pl9;24hours later the expression of RANKL and IL-17R mRNA was detected by real time-PCR, and RANKL protein was measured by western blot.Results:The level of RANKL mRNA and protein were upregulated by IL-23p19in various concentrations. The most effective dose in inducing RANKL expression was5ng/ml of IL-23pl9, and the most effective time was for24h. IL-23pl9, IL-17A, IL-17F, IL-23p19jointed with IL-17A and IL-23p19jointed with IL-17A all could induce the mRNA expression of RANKL. IL-23p19, IL-17A and IL-17F could induce the expression of IL-17R. The level of IL-17R was upregulated after the stimulation of inhibitor of NF-κB, inhibitor of Erk and inhibitor of JAK2. Inhibitor of JNK, inhibitor of PI3K could induce the expression of RANKL in human FLS; however, the level of RANKL was down-regulated by inhibitor of JAK2, inhibitor of JAK3, inhibitor of NF-κB, inhibitor of Erk.Conclusion:IL-23p19could increase the expression of RANKL mRNA and protein in RA-FLS. IL-23p19and IL-17both can induce the expression of RANKL and IL-17R. The upregulation of of IL-23p19-induced-IL-17R was mediated by NF-κB、Erk、JAK2. IL-23p19-induced RANKL expression by FLS was mediated by NF-κB、JAK2, JAK3and Erk.Summary1. FLS from RA patients can be successfully and effectively cultured by means of tissue culture.2. IL-23p19could upregulate the expression of RANKL mRNA and protein.3. IL-23p19could upregulate the expression of RANKL by NF-κB、JAK2, JAK3and Erk.4. IL-23p19and IL-17could induce the expression of RANKL and IL-17R.5. IL-23p19could upregulate the expression of IL-17R by NF-κB、Erk、JAK2.