| Background and purposeImmune thrombocytopenia(ITP)is a kind of acquired autoimmune disease.The pathogenesis of ITP has not been fully clarified up to now,in recent years,some scholars have proposed that the imbalance of Th1 and Th2 ratio and the abnormal number and function of Treg cells are closely related to the occurrence of ITP.However,the changes of the number and functional defects of CD4~~+Tregs and CD8~~+Tregs have not been uniformly reported in ITP patients with different courses.This study mainly explored the expression changes of Th1,Th2,CD4~~+Tregs,CD8~~+Tregs in ITP patients with different courses(newly diagnosed,persistent and chronic),and analyzed their relationship with the courses of ITP,aims to provide a basis for the immunotherapy of ITP.Materials and Methods(1)Research object70 patients with ITP who treated in our hospital from December 2019 to December 2020 were selected,they were divided into the new diagnosis group(28cases,patients within 3 months after diagnosis),the persistent group(20 cases,patients with persistent thrombocytopenia 3 to 12 months after diagnosis)and the chronic group(22 cases,patients with continuous reduction of platelets for more than12 months)according to the diagnostic criteria of different courses in the"Chinese Guidelines for the Diagnosis and Treatment of Primary Immune Thrombocytopenia in Adults(2016 Edition)".Another 25 healthy people who received physical examination during the same period were selected as the control group.(2)Flow cytometry to detect the percentage of Th1 and Th2 cell subsets in PBMCsPeripheral blood mononuclear cells(PBMCs)in each group were separated by density gradient centrifugation of lymphocyte separation solution.Diluted PBMCs and RPMI 1640 1:1 in equal volume,added PMA/Lonomycin and Brefeldin A,incubated for 4-6 hours;took the cell suspension into the flow tube,and added APC-CD3,PC5.5 and Fixation Buffer respectively,Permeabilization Buffe,incubated for 15 min,centrifuged and discarded the supernatant,added FITC-IFN-γ/PE-IL-4and isotype control respectively,detected by flow cytometer after sample processing.(3)ELISA to detect the serum levels of multiple cytokines of Th1 and Th2 cellsThe serum was separated after centrifugation of the blood sample,the ELISA plate was coated with IL-2,IL-10,IFN-γand IL-4,the serum and monoclonal antibody were added to incubate,washed repeatedly with PBS,than horseradish peroxidase goat anti-mouse Ig G antibody was added,using o-phenylenediamine as the color developing agent,measure the respective absorbance values on the microplate reader after color development.(4)Flow cytometry to detect the percentage of CD4~~+Tregs and CD8~~+Tregs subgroups in PBMCsTook two flow tubes,added 100μL of the prepared PBMCs respectively,and added the antibody APC-CD3/FITC-CD4/PE-CD25/PC7-Foxp3 to the CD4~~+Tregs flow cytometer;and added the antibody APC-CD3/PC5.5-CD8/PE-CD28 to the CD8~~+Tregs flow cytometer;set isotype control;detected by flow cytometer after sample processing.(5)Statistical methodsSPSS 20.0 was used for data statistics,measurement data was expressed as mean±standard deviation(x±s),using independent sample t test;counting data was expressed as percentage(%),usingχ~2 test;the correlation used Pearson correlation analysis,the difference was statistically significant with P<0.05.Results(1)Compared with the control group,the percentage of Th1 cells in PBMCs of patients in the new diagnosed group,the continuous group and the chronic group gradually increased,the percentage of Th2 cells gradually decreased,and the pairwise comparisons between groups were statistically different(P<0.05).(2)Compared with the control group,the serum levels of Th1 cytokines(IL-2,IFN-γ)in the new diagnosed group,the continuous group and the chronic group gradually increased,the levels of Th2 cytokines(IL-4,IL-10)gradually decreased,and the pairwise comparisons between groups were statistically different(P<0.05).(3)Compared with the control group,the percentages of CD4~~+CD25~~+Foxp3~~+Tregs in CD4~~+T cell and CD8~~+CD28~-Tregs in CD8~~+T cell in the new diagnosed group,the continuous group and the chronic group gradually decreased,and the pairwise comparisons between groups were statistically different(P<0.05).(4)The Pearson correlation analysis showed that Th1 cells were positively correlated with the course of ITP(r=0.464,P=0.020),while Th2 cells,CD4~~+CD25~~+Foxp3~~+Tregs and CD8~~+CD28~-Tregs were negatively correlated with the course of ITP(r=-0.357,P=0.016;r=-0.728,P=0.010;r=-0.223,P=0.034).ConclusionTh1 cells are positively correlated with the course of ITP,the longer the course of the disease,the more serious of Th1/Th2 drifts to Th1;Th2 cells,CD4~~+CD25~~+Foxp3~~+Tregs and CD8~~+CD28~-Tregs are negatively correlated with the course of ITP,the longer the course of the disease,the down-regulation of Th2 cells,CD4~~+CD25~~+Foxp3~~+Tregs and CD8~~+CD28~-Tregs are more significantly. |
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