The Dynamic Study Of Th1/Th2Cell And Their Cytokines In Collagen Induced Arthritis Mouse Model

Objective:RA(rheumatoid arthritis,RA) is a chronic systemic inflammatoryautoimmune disease,whose main manifestation is peripheral joints synovitisand gradually induced cartilage destruction and bone erosion at differentdegree，which resulting in joint deformity and stiffness at last.70%～80%ofpatients eventually develop into different degrees of barrier function, andeven disability. Currently,the number of RA patients is up to more than400million in China, so it is one of the main causes of losting labour ability. Thepathogenesis of RA is unclear,infection, environmental, genetic and immunefactors may play important roles in the occurrence and development of RA.With recent progress in immunology,some studies indicated:CD4+T cellsubsets Th1/Th2are involved in all progess of RA and playing important rolesin the pathogenesis of RA,but the results are different. So we choosecollagen-induced arthritis (CIA) as mouse model to observe the dynamicchanges of Thl and Th2subsets in the CIA,and to explore deeply the effects ofTh1/Th2cells and their cytokines in the pathogenesis of RA. So our researchwill provide furthe expermental basement to treatment of RA.Methods:1Two batch of SPF male DBA1/J mice,90per batch, including42asnormal control group, the rest of the48as CIA modle group.Each batch micewere randomly divided into seven time points (7d,14d,21d,28d,35d,42d and56d after primary immunization),normal control group（n=6）,CIA modelgroup（n=6）at each time point.2The mice were respectively extracted the eyeball for blood andseparated the serum Under the sterile condition on the7d,14d,21d,35d,42d and56d after primary immunization. Then the dynamic changes of cytokinesof Th1/Th2cell in different periods were detected by flow cytometry.3The mice were killed after blooding,then the spleen be taken and weremade into single cell suspension to detect the dynamic changes of Th1/Th2cell by flow cytometry at seven time points.4The mice were killed after blooding,then the spleen be taken and weremade into single cell suspension to detect the expression changes of Th1/Th2cell specific transcription factor T-bet/GATA by Real-time quantitative-PCRat seven time points.5Use SPSS17.0statistical software to analyze the above mentioned data.Results are presented as Mean±SE. Differences between groups were analyzedusing t-test by ANOVA.α=0.05is the standard of significant test.RESULTS:1The dynamic changes of Th1/Th2cell by flow cytometryCompared with normal control group, the percentage of spleen Th1cellsin CIA mice was significantly increased on the7d and14d after primaryimmunization (P<0.01), however，there was no significant difference on the21d(P>0.05),until the28d it was significantly increased (P<0.01)，then beganto decline to the normal group level on the35d(P>0.05) and continue todecline on the42d (P<0.01), there was no significant difference between themodel group and the control group on the56d (P>0.05).Compared with normal control group, the percentage of spleen Th2cellsin CIA mice were significantly increased on the7d and14d after primaryimmunization (P<0.01), on the21d and28d there was no significantdifference compared with normal control group (P>0.05),it began to increasesignificantly than that of normal control group level on the35d and42d(P<0.01), while on the56d significantly decline compared with the normalcontrol group (P <0.05).2The expression changes of Th1/Th2cell specific transcription factorT-bet/GATA by Real-time quantitative-PCRCompared with normal control group, the level of spleen T-bet expression in CIA mice was significantly increased7d and14d after primary immune(P<0.05), and on the21d decreased compared with the normal group, therewas no significant difference (P>0.05),but T-bet expression began to rise onthe28d (P<0.05).On the35d T-bet expression compared with the normalgroup, there was no significant difference (P>0.05), and began to decline onthe42d (P<0.05)and there was no significant difference between themodel group and the control group on the56d (P>0.05).Compared with normal control group,the level of spleen GATAexpression in CIA mice had no significant difference between the model groupand the control group on the7d after primary immunization (P>0.05), butbegan to increased on the14d (P<0.05), on the21d and28d GATA expressdecline into normal control group level (P>0.05), until the35d,42d and56dGATA expression had increasing tendency and there was significant differencecompared with that of normal group (P<0.05).3The dynamic changes of Th1/Th2cytokines by flow cytometry3.1The results of dynamic changes of Th1cytokinesCompared with normal control group,the levels of IFN-γ has no statisticalsignificance on the7d after primary immunization (P>0.05), however,on the14d it was significantly higher than normal control group(P<0.01)，there is no statistical significance on the21d (P>0.05).On the28dIFN-γ is significantly higher than normal group and reached the peak (P<0.01),then the35d IFN-γ was decreased, but still higher than normal group(P<0.05)and it gradually decreased to normal level on the42d and56d (P>0.05).Compared with normal control group,the level of TNF-α was increasedobviously on the7d and14d after primary immunization (P<0.05), there wasno statistical significance compared with that of normal control group21d(P>0.05), untill the28d and35d after primary immune TNF-α increasedsignificantly (P<0.05), it increased more significantly on the28d (P<0.01).Onthe42d and56d TNF-α had no statistical significance (P>0.05).3.2The results of dynamic changes of Th2cytokines Compared with normal control group,the level of IL-4on the7d,14d afterprimary immunization were higher than that of normal control group and thedifference was statistically significant (P<0.05), it decreased to normal leveon the21d (P>0.05).Untill the28d IL-4was significantly higher than that ofnormal control group (P<0.01), but the35d,42d and56d had no statisticalsignificance (P>0.05).Compared with normal control group，the level of IL-5on the7d afterprimary immunization was significantly increased than that of the normalcontrol group and the difference was statistically significant (P<0.05), but ithad no statistical significance on the14d,21d,35d,42d and56d (P>0.05).Conclusion:1CD4+Th1/Th2cell and their cytokines participate in the pathogenesisof CIA in the different stages and have regular relation to development ofRA,the changes of balance between them are closely related to the degree ofdisease activity.2Detection of Th1/Th2cells and their cytokines help determine theprogression of patients with RA.