| AIHA/ES is a form of immune-related cytopenia caused by autoantibodies that target the antigens on the red blood cells.B-lymphocyte synthetize and secrete autoantibodies and B-lymphocytes have an important role in the pathogenesis of AIHA/ES.Better understanding of AIHA can provide a theoretical basis for potential targets of novel therapies.Mi R-150 is selectively expressed in mature or resting B-lymphocytes and normally highly expressed in mature B-lymphocytes.The study on the regulation of miR-150 is helpful in understanding the pathogenesis of many autoimmune diseases.CD69,a C-type lectin,is induced early following activation of lymphocytes.We investigated the expression of T cells and CD69 in AIHA/ES.Our study also explores the expression of Th17 cells to analyze the role of regulatory T cells in the pathogenesis of AIHA/ES.Part1 The expressions of miR-150 and c-Myb and their clinical significances in autoimmune hemolytic anemiaObjective: To study the expression of B lymphocytes in patients with autoimmune hemolytic anemia(AIHA)/Evans syndrome and investigate the relevance between the expression of miR-150 and c-Myb to understand the regulation of B lymphocytes in patients with(AIHA)/Evans syndrome.Methods: 35 patients with AIHA / Evans(18 hemolytic episode patients,17 remission patients),15 healthy controls(NC)and 6 chronic lymphocytic leukemia(CLL)patients were enrolled from October 2015 to May 2016 into this study in Department of Hematology,General Hospital of Tianjin Medical University.The quantity and the percentage of total circulating B-lymphocytes in patients were analyzed by FCM.CD19~+ lymphocytes from Peripheral blood(PB)were sorted by magnetic activated cell sorting(MACS).C-Myb m RNA and miR-150 were analyzed by real-time PCR(RT-PCR).Correlation between c-Myb and miR-150 and clinical parameters of AIHA/Evans patients was analysed.Results:1、 The percentage and the quantity of B-lymphocytes in hemolysis group was(19.35±16.20)% and(29.73±19.92)×107/L respectively,which were significantly higher than those of remission group patients [(6.58±4.43)%(10.96 ± 7.98)×107/L] and normal controls [(6.69 ± 2.12)%,(9.92 ± 4.03)× 107/L](P <0.05).There were no significant differences in the percentage and the quantity of B-lymphocytes between the latter two groups.The percentage and quantity of B-lymphocytes in CLL patients were(70.42±20.15)% and(1503.40±1184.78)×107/L,significantly higher than those of AIHA/ES patients and normal controls(P < 0.05).2 、 C-Myb m RNA in hemolytic episode patients was(17.65 ± 14.91),significantly higher than those of remission patients(9.23±8.22,P<0.05),NC(3.93±6.37,P<0.01)and CLL patients(7.36±5.16,P<0.05).There was no statistical difference among remission patients,NC and CLL patients.3、Patient’s c-Myb m RNA was negatively correlated with their hemoglobin(Hb)and complement 3(C3)(P<0.05),positively correlated with total bilirubin(TBIL)and indirect bilirubin(IBIL)(P<0.05).4、miR-150 of hemolytic episode patients was(0.36±0.49),significantly lower than those of remission patients(0.76±0.73,P<0.05),NC(1.24±0.73,P<0.01)and CLL patients(1.79±1.30,P<0.01).5、miR-150 was negatively correlated with TBIL,IBIL(P<0.05)and positively correlated with Hb,C3(P<0.05).6、C-Myb m RNA in hemolytic episode patients and remission patients was respectively negatively correlated with miR-150.Conclusions: The percentage of B-lymphocytes increased in AIHA/Evans syndrome patients.C-Myb,a regulation factor of B lymphocyte,increased and miR-150 decreased in AIHA/Evans patients.C-Myb and miR-150 were inversely correlated and they were associated with the severity of AIHA / Evans syndrome.Part2 The expression of Th17 cells on T cells in autoimmune hemolytic anemia Objective:To explore the roles of Th17 cells in the pathogenesis of autoimmune hemolytic anemia.Methods: 24 patients with AIHA/Evans(14 hemolytic episode patients,10 remission patients),10 healthy controls.The percentage of Th17(CD3~+CD4~+IL-17+)and CD3~+CD4~+IL-17+/CD3~+ were detected by flow cytometry.Results: 1、The CD3~+CD4~+IL-17+/CD3~+CD4~+ lymphocytes ratio in hemolytic episode group(3.34±1.9)% was higher than that in remission group(1.59±0.70)%(P<0.05)and healthy group(1.58±0.99)%,(P<0.05).There was no statistical difference between the remission group and healthy group.2、The CD3~+CD4~+IL-17+/CD3~+T lymphocytes ratio in hemolytic episode group(1.66±1.04)% was higher than that in remission group(0.97±0.50)%(P<0.05)and healthy group(0.87±0.48)%,(P<0.05).There was no statistical difference between the remission group and healthy group.Conclusions: This study proved that the percentage CD3~+CD4~+IL-17+/CD3~+CD4~+ and CD3~+CD4~+IL-17+/CD3~+T lymphocytes ratio increased in AIHA/Evans syndrome patients.Part3 The expression of CD69 on CD4~+ T cells in autoimmune hemolytic anemiaObjective: To explore the expression of the T cell subsets and CD69 on CD4~+ T cell and their mechanism in autoimmune hemolytic anemia.Methods: 32 patients with AIHA/Evans(15 hemolytic episode patients,17 remission patients),13 healthy controls.CD4~+T lymphocytes from peripheral blood(PB)were sorted by magnetic activated cell sorting(MACs).The percentage of T cell subsets and CD69 on CD4~+ T cell were analyzed by flow cytometry.Also,the expression of CD69 on CD4~+ T cells was detected using real-time PCR(RT-PCR).Correlation between CD69 and clinical parameters of AIHA/Evans patients were analysed.Results:1 、 The CD3~+CD4~+/CD3~+、 CD3~+CD8~+/CD3~+T lymphocytes ratio and the CD3~+CD4~+、 CD3~+CD8~+T lymphocytes count in three groups did not differ significantly.The ratio of CD4~+/CD8~+ in hemolytic episode group(1.33±0.73)was higher than that in healthy group(0.88±0.32,P<0.05).2、In hemolytic episode patients,the ratio of CD3~+CD69~+/CD3~+T lymphoc ytes[(3.08±1.48)%] was significantly higher than that in healthy controls[(1.28±0.83)%,P<0.01] and in remission group[(1.96±1.33)%,P<0.05].The quantity of CD3~+CD69~+/CD3~+T lymphocytes in hemolytic episode group [(2.94±1.81)×107/L] was significantly higher than that in healthy controls[(1.48±1.42)×107/L,P<0.05].3、The percentage of CD3~+CD4~+CD69~+/CD3~+CD4~+T cells in hemolytic episode group [(2.16±1.56)%] was significantly higher than that in remission group [(1.16±0.62)%,P<0.05] and healthy controls[(0.94±0.78)%,P<0.05].The quantity of CD3~+CD4~+CD69~+T lymphocytes in hemolytic episode group[(1.04±0.98)×107/L] was higher than that in healthy controls[(0.44±0.38)×107/L,P<0.05].4、The percentage of CD3~+CD8~+CD69~+/CD3~+CD8~+T cells in hemolytic episode group [(4.87±2.56)%] was significantly higher than that in healthy controls[(1.83±1.27)%,P<0.01].The quantity of CD3~+CD8~+CD69~+T lymphocytes in three groups showed no significant difference.5、The ratio of CD3~+CD4~+CD69~+/CD3~+CD4~+T lymphocytes in hemolytic episode group was negatively correlated with hemoglobin(Hb)(P < 0.01),positively correlated with the percentage of reticulocyte(Ret%)(P=0.01)total bilirubin(TBIL),indirect bilirubin(IBIL)(P<0.01)and not correlated with Ret count,lactic dehydrogenase(LDH),complement 3(C3),complement 4(C4).6、The ratio of CD3~+CD4~+CD69~+/CD3~+CD4~+T lymphocytes in remission group was negatively correlated with Hb(P<0.05).There were no correlations between CD3~+CD4~+CD69~+/CD3~+CD4~+T lymphocytes and Ret count,LDH,TBIL,IBIL,C3,C4 in remission group.7、CD69 m RNA in hemolytic episode patients was(32.26±35.11),significantly higher than that in remission group(6.05±5.87)(P<0.05),healthy controls(1.76±1.85)(P<0.01).CD69 m RNA in remission group was significantly higher than healthy controls(P<0.05).8 、 The level of CD69 in serum in hemolytic episode patients [(494.21±16.06)pg/ml] was significantly higher than that in healthy controls [(441.39±104.6)pg/ml,P<0.05].Conclusions: This study proved that the CD69 in CD4~+T lymphocytes increases expression levels in AIHA/Evans patients and is associated with the severity of the AIHA/Evans,which provide a theoretical basis for the role of T cells in pathogenesis of AIHA/Evans. |
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