| Rheumatoid arthritis is a kind of chronic autoimmune disease with the joints inflammation characterized by a large number of T lymphocytes infiltration in the synovial. To now, the pathogenic mechanism in RA is also unclear. However that the unknown antigen is presented to T helper (Th) cells is at the hub in the pathogenesis.There are a lot of data that suggest Th types and cytokines contribute to the pathogenesis in RA. Naive conventional CD4 T cells have open to their 4 distinct fates that are determined by the pattern of signals during their initial interaction with antigen.These 4 population are Thl,Th2, Th17 and regulatory T (Treg) cells. They exert such functions mainly through secreting cytokines and chemokines that play different functions. Th cells play a central role in cellular defence against pathogens as well as in cellular self-tolerance. The activation of Th cells is a crucial process determining the course of a protective immune response. Dysregulated activation processes can lead to pathologic immune reactions and may induce autoimmune diseases.Th cells are at the hub of this paradigm, which was developed in the 1980s. Many studies led to the belief that RA was a Thl-driven disease. The Thl phenotype is associated with inflammation in RA, whereas the Th2 phenotype copes with inflammation to some extent. The excess of pro-inflammatory cytokines and relative deficiency in anti-inflammatory cytokines define the Thl/Th2 imbalance, which was believed to drive RA. The Th17 cells increase the process of inflammation and joints destruction in RA. Treg cells play a critical role in competing with inflammation and maintaining self-tolerance as well as regulating immune responses. The study belive that Th17 cells and Treg cells play a important role, especially in the latter process. Although Treg cells play a critical role in the pathogenesis in RA, the imbalance of Thl/Th2 and Thl7/Treg may be the pathogenic mechanism of RA. It has been speculated that Thl7/Treg cells may be involved in the occurrence of disease and development as Thl/Th2 cells. Given Thl, Th2, Thl7 cells and Treg cells are differentiated from CD4+T cell, it is a very complex network which is mutuall interaction and inhibition in the four phenotype cells. The new paradigm must be further confirmed. But the speculation haven't been studied by our or foreign workers. To further explore the action of the Th1,Th2, Th17 and regulatory T cells to rheumatoid arthritis, we have the study in cell, protein and gene level.ObjectiveTo investigate the effect of lymphocyte apoptosis, Thl cells,Th2 cells, Th17 cells, Treg cells and related cytokines in RA.The study detected the number and function of the four phenotype cells and the expression of transcription factor subtypes by detecting the peripheral blood lymphocytes,blood serum and lymphocytic mRNA in patients with RA.MethodsFirstly,all the RA patients selected into this study were divided into different disease activity groups according to Disease Activity Score.The 3H-TdR was added to the peripheral blood lymphocytes for 24 hours and then filtered,fixed, dried and measured the decay of values per minute using liquid scintillation counting. At the same time the lymphocytes were added into the dual-layer agarose gel, cracked, unwinded, electrophored and stained. Analysis CASP comet image automatic analysis software illustrated the relationship between lymphocytes metabolism and the disease preliminarily. The lymphocytes were marked by CD4, PI, AnnexinⅤand then detected apoptosis of CD4+lymphocyte by flow cytometry to analysis the relationship between the CD4+lymphocyte apoptosis and diseases in RA furtherly.The peripheral blood mononuclear cells were cultured, stimulated, as well as in vivo markered and then detected the number of the Thl phenotype cells, Th2 phenotype cells, Th17 phenotype cells and Treg phenotypes cell number to analysis the relationship between the various subtypes of Th cell and disease in RA furtherly. The relevant levels of cytokines in peripheral blood were found through the Cytometric Bead Array and Enzyme-linked Immunosorbent Assay to detect the relationship between function of the various subtypes of Th cell and the desease in RA. Finally, The RNA in Peripheral blood mononuclear cells were extracted and measured expression of transcription factors of the four phenotypes cells by Real Time PCR. All data were expressed as Mean±standard deviation (x±s) or median±quartile range(M±QR). The difference between different groups was assessed with t-test or Wilcoxon-test and Kruskal-Wallis H-test. If the pvalue was less than 0.05 or 0.0083,It was considered to be a statistically significant difference.Results1. The DNA metabolic activity of peripheral lymphocyte was higher in RA compared with the control, but there was no statistical significance (P> 0.05); The DNA damage of peripheral lymphocytes in RA was higher compared with the control,but there was no statistical significance (P> 0.05); The apoptosis of CD4+T lymphocyte was lower than the control,but there was no statistical significance (P<0.05).2. The percentage of Thl cells in peripheral lymphocyte in RA was higher than that in the control, but there was statistical significance (P<0.05); The percentage of Th2 cells in peripheral lymphocyte in RA was higher than that in the control, but there was no statistical significance (P>0.05); The percentage of Thl7 cells in peripheral lymphocyte in RA was higher than that in the control, but there was statistical significance (P<0.05); The percentage of Treg cells in peripheral lymphocyte in RA was higher than that in the control, but there was statistical significance (P<0.05);3. The percentage of Thl and Th2 cells in peripheral lymphocyte in RA with DAS28≤3.2 was higher than that in the control, but there was no statistical significance (P>0.05), The percentage of Th17 cells in peripheral lymphocyte in RA was higher than that in norma and there was statistical significance (P<0.05), The percentage of Treg cells in peripheral lymphocyte in RA was lower than that in the control and there was no statistical significance (P>0.05); The percentage of Thl and Th2 cells in peripheral lymphocyte in RA with 3.2 0.05), The percentage of Th17 cells in peripheral lymphocyte in RA was higher than that in the control and there was statistical significance (P<0.05), The percentage of Treg cells in peripheral lymphocyte in RA was lower than that in the control, but there was no statistical significance (P>0.05); The percentage of Thl and Thl7 cells in peripheral lymphocyte in RA with DAS28>5.1 was higher than that in the control and there was statistical significance (P <0.05); The percentage of Treg cells in peripheral lymphocyte in RA was lower than that in the control and there was statistical significance (P>0.05),The percentage of Th2 cells in peripheral lymphocyte in RA was higher than that in the control, but there was no statistical significance (p>0.05).4. The serum concentration of IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-y and TNF-a in RA were increased compared with the control,and there was statistically significant (p<0.05); The level of TGF-βwas also increased compared with the control,but there was no statistical significance (P> 0.05).5. The serum concentration of IL-2, IL-4 and TNF-a in RA with DAS28≤3.2 was increased compared with the control, and there was statistical significance (P<0.0083); The serum concentration of IL-6, IL-10 and IL-17A was greater than the control,but there was no statistical significance (p> 0.0083);The serum concentration of IFN-γand TGF-βwas lower than control,but there was no statistical significance (p> 0.0083); The serum concentration of IL-2, IL-6, IL-10 and IL-17A in RA with 3.2 0.0083);The serum concentration of IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-y and TNF-a in RA patients with DAS28> 5.1 was increasd compared with the control,and there was statistical significance (p＜0.0083); Althoght the serum concentration of TGF-P was higher compared with the control,but there was no statistical significance (P> 0.0083). 6. The expression level of T-bet, ROR-yt, GATA-3, Foxp3 mRNA that is dividely specific transcription factor of Thl, Th2, Th17 and Treg cells in RA is 17.01,26.12,28.31,29.11.Conclusion1. The proliferation CD4+T lymphocytes in RA patients are abnormal.It may be due to obstruction caused by apoptosis.2. The Th17/Treg cells play an important role in RA as Thl/Th2 cells. They may be involved in different stages of occurrence and development in the disease.3. TheTh cells secrete cytokines to promote pro-inflammatory cytokines and anti-inflammatory factor in the imbalance, which participate in the pathogenesis of RA.4. The Thl, Th2 and Th17 cells work by secreting cytokines, while Treg cells cope with the inflammation through direct contact between cells.5. The T-bet, ROR-yt, GATA-3 and Foxp3 mRNA which are special transcription factor of the Thl, Th2,Th17 and Treg cells are expressed respectively in RA patients. |
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