The Study Of Serum Cytokine And PBMC MRNA Profiles In Patients Of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a systemic, inflammatory autoimmune disorder that presents as a symmetric polyarthritis associated with swelling and pain in multiple joints, often initially occurring in the joints of the hands and feet. Articular inflammation causes activation and proliferation of the synovial lining, expression of inflammatory cytokines, chemokine-mediated recruitment of additional inflammatory cells, as well as B cell activation with autoantibody production. A vicious cycle of altered cytokine and signal transduction pathways and inhibition of programmed cell death contribute to synoviocyte and osteoclast mediated cartilage and bone destruction. In the last few years, the study of serum cytokine profiles and cytokine mRNA expressions in PBMC in patients of RA have become a hot spot in the pathogenesis of RA.The study was divided into two parts. In the first part, this study was designed to investigate the mRNA expression and protein level of cytokines in PBMCs stimulated with CD3 mAb, and to compare the pattern of cytokine protein and cytokine mRNA elevations in PBMCs with that of controls. The PBMCs of healthy adult were isolated by Ficoll and incubated with anti-CD3 mAb coated microwell plates. Cytokine levels of IL-2,IFN-γand IL-10 in culture supernatant were tested by ELISA. PBMCs stimulated by CD3 mAb were selected to express mRNA of cytokines. Cytokine gene fragments of were amplified using specific primers. PCR products were inserted into the PMD18-T vector. Recombinant plasmids were selected after sequencing. Target genes and house keeping gene used asβ-Actin were amplified on fluorescence quantitative PCR. Standard amplification curves were developed at the concentration of target genes andβ-Actin ranging from 103,104,105 and 106 copies/ml. The results showed that: 1. Standard amplification curves were established with high specificity, reliability and stability. 2. The expression of mRNA in PBMCs stimulated by CD3 mAb was significantly higher than that of controls. 3. The levels of in supernatant showed significant difference between the PBMCs stimulated with CD3 mAb and controls. Compared with controls, mean cytokine levels of IL-2,IFN-γand IL-10 were all higher in CD3 mAb co-cultured group. 4. Cytokine levels of IL-2,IFN-γand IL-10 have great relation with the mRNA expression of cytokine in PBMCs.In the second part, we continuingly established standard amplification curves of IL-1β,IL-6,IL-8 and TNF-α. The study was proposed to investigate cytokine mRNA expression in PBMCs and protein level of cytokines in active RA patients, and to compare the pattern of cytokine protein and cytokine mRNA elevations in active RA patients with that of stable RA patients and healthy controls. We also explore the functions of molecules expressed on CD4+T cells inducing activation of cells and apoptosis in causing imbalanced immunity of patients with RA. The results showed that: 1. Expression of CD154 significantly increased on CD4+T cells in patients with active RA, comparing with stable RA and healthy controls. 2. Soluble CD154 in plasma with active RA was higher than that of stable RA patients and controls. 3. Compared with controls and stable RA patients, mean serum levels of IL-1β, IL-6, IL-8, IL-10 and IFN-γwere all higher in patients with active RA. In contrast, stable RA patients show higher level of IL-10 compared with controls. 4. Basal levels of cytokine mRNA expression in PBMCs of controls were different. mRNA expression of IL-6, IFN-γ, IL-10 and TNF-αshowed a more general increase in patients with active RA comparing with controls and stable RA patients.In summary, mRNA expression of cytokines in PBMCs could be measured by fluorescence quantitative PCR. Both the levels of cytokine in serum and mRNA cytokine expression in PBMCs were significantly higher in patients with active RA than that of stable RA patients and controls. All these results showed the measurement of mRNA expression in PBMCs and serum level of cytokines in patients with active might play an important role in RA diagnosis and prognosis in clinic.