| BackgroundRheumatoid arthritis(RA)is one of the most common autoimmune rheumatic diseases,it is mainly through the several complex systems of inflammatory factors to affect the joint function.RA patients will have joint deformities,mutilation and rheumatoid coronary heart disease,if not be treated timely.RA pathogenesis is not clear at present,scholars have considered that it has relationship with genetic factors,environmental factors,viral infections and autoimmune disorders.Some factors cause the body's natural immune and adaptive immune response,it leads to a further joint damage.The disordered balance of regulatory T cells/helper T 17 cells has an important role in the development of RA disease.The proportion of Treg cells and the secretion of IL-10 cytokines in the peripheral blood of RA patients decreased.Treg cells can affect a variety of cell functions,such as monocytes,dendritic cells,B cells,etc.B cells in RA patients appear high activation state,secrete several IgM and IgG antibodies,these antibodies are called rheumatoid factors.This paper intends to use flow cytometry and ELISA technique to analysis of the relationship between the percentage of Treg cells,IL-10,IgM and IgG in peripheral blood of RA patients,and then investigate the effects of IL-10 on the differentiation of B cells in vitro.ObjectiveThis paper intends to uncover the proportion of Treg cells,IL-10,IgM and IgG in RA patients through experiment;To prove IL-10 secreted by Treg can inhibit the differentiation of B cells and autoantibodies secretion in RA patients,and then reveal the lack of regulatory T cells can not inhibit IL-10 in RA patients,and also provide theoretical basis for RA treatment and important targets.Methods1.Subjects investigated: Referring to The American society for rheumatism in 1987,20 patients with RA were enrolled in our hospital.The control group was 20 cases of healthy people in the hospital during the same period with the age and gender matched.2.The collection and processing of samples: Peripheral blood mononuclear cells were collected from HC and RA.The separated plasma are stored in-80?refrigerator for ELISA.3.Detection the level of plasma IL-10: using human IL-10 ELISA kit to detect the level of plasma IL-10.4.Detection the level of plasma IgM?IgG: using human IgM?IgG ELISA kit to detect the level of plasma IgM?IgG.5.Detection of CD4~+CD25~+Foxp3~+Treg cell proportion: Detection and analysis of CD4~+CD25~+Foxp3~+Treg cell proportion in samples by flow cytometry.6.Treg cells inhibit B cell differentiation: after CD4~+CD25~+ T cells and naive B cells training together,detection and analysis of CD19~+IgD~-CD38~+CD27~+plasma cell proportion and IgM?IgG mRNA expression level in samples by flow cytometry.7.IL-10 antibody blocking Treg cells inhibit B cell differentiation: add IL-10 antibody into the co-culture of CD4~+CD25~+T cells and naive B cell,detection and analysis of CD19 + IgD~-CD38~+ CD27~+ plasma cell proportion and IgM?IgG m RNA expression level.8.Measurement data with standard deviation of mean and subtract,compare the difference between the two groups using t test,the data were compared by chi-square test,correlation using Pearson correlation analysis.Inspection level of alpha = 0.05,P < 0.05 for the difference was statistically.Results1.Specimen grouping situation: RA group a total of 20 cases,HC group a total of 20 cases,there is no statistical difference between gender and age.2.The results of automatic biochemical analyzer: the plasma IL-10 content in patients with RA is(32.04±5.96)pg/ml,that in healthy controls is(57.68±13.72)pg/ml,the former was lower than the latter,and there was a statistical difference.3.The results of automatic biochemical analyzer: the plasma IgM content in patients with RA is(23.25±7.85)IU/ml,higher than that in healthy controls(13.63±3.44)IU/ml,the plasma IgG content in patients with RA is(26.89±8.67)IU/ml,higher than that in healthy controls(13.39±3.96)IU/ml,and both of them have a statistical difference.4.The plasma CD4~+CD25~+Foxp3~+T cell proportion in RA patients is(5.04 + 1.58)%,and in control group is(9.86 + 3.89)%,the former was lower than the latter,and there was a statistically significant.5.Before adding Treg cells,the proportion of plasma cells is(4.31 + 2.51)%,after adding Treg cells,the proportion of plasma cells is(1.83 + 1.23)%,which is lower than the former,and there is a statistical difference;not adding the Treg cells,the expression of IgM and IgG mRNA,is respectively(2.01-0.57)and(1.97 + 0.52)times,these were higher than adding Treg cells,and there is a statistically significant.6.Before adding the IL-10 antibodies,the proportion of plasma cells is(1.83 + 1.23)%,after adding the IL-10 antibodies,the proportion of plasma cells is(4.73 + /-1.95),the latter is significantly higher than the former and statistically significant;after adding the IL-10 antibodies,the expression of Ig M and IgG mRNA is respectively(2.17 + 0.80)and(2.08 + 0.85)times,these are higher than no IL-10 antibodies group,and there is a statistically significant.Conclusions1.Treg cells and IL-10 content reduced,IgM and IgG content increased in RA patients with peripheral blood.2.Treg cell can restrain the differentiation of B cells in patients with RA,and suppress the secretion of autoimmune antibody,it is revealed that the decrease of Treg cells can not inhibit the secretion of autoimmune antibody,because of IL-10 deficiency. |
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