原发性免疫性血小板减少症(ITP)患者CD5^+B细胞对CD4^+T细胞Th1/Th2型细胞因子分泌的影响  

Impaired ability of CD5^+B cells to regulate Th1/Th2 cytokines secretion by CD4^+T cells in patients with primary immune thrombocytopenia(ITP)

作　　者：于慧辉 詹延霞 季丽莉 李炀 孙丽华 程韵枫 化范例 YU Hui-hui;ZHAN Yan-xia;JI Li-li;LI Yang;SUN Li-hua;CHENG Yun-feng;HUA Fan-li(Department of Hematology,Jinshan Hospital,Fudan University,Shanghai 201508,China;Department of Hematology,Qingpu Branch of Zhongshan Hospital,Fudan University,Shanghai 201700,China;Department of Hematology,Zhongshan Hospital,Fudan University,Shanghai 200032,China)

机构地区：[1]复旦大学附属金山医院血液科,上海201508 [2]复旦大学附属中山医院青浦分院血液科,上海201700 [3]复旦大学附属中山医院血液科,上海200032

出　　处：《复旦学报（医学版）》2020年第1期53-58,共6页Fudan University Journal of Medical Sciences

基　　金：上海市自然科学基金(19ZR1410000);上海市科委引导类项目(19411972200);上海市卫计委基金(201840352)~~

摘　　要：目的探讨原发性免疫性血小板减少症(immune thrombocytopenia,ITP)患者CD5+B细胞对CD4+T细胞分泌Th1/Th2型细胞因子的调节作用及其在大剂量地塞米松(high dose dexamethasone,HD-DXM)治疗后的变化。方法采集5例初发原发性ITP患者HD-DXM治疗前后外周静脉血各20 mL及5例健康对照者外周静脉血20 mL,Ficoll密度梯度离心法分离外周血单个核细胞(peripheral blood mononuclear cell,PBMC),进一步以免疫磁珠法分选CD5+B细胞、CD5-B细胞和CD4+T细胞。CD5+B细胞或CD5-B细胞与CD4+T细胞按1∶9、1∶5、1∶3、1∶1、3∶1的梯度比例混合培养72 h或144 h。ELISA方法检测细胞培养上清液中IFN-γ(代表Th1型细胞因子)和IL-4(代表Th2型细胞因子)水平。结果共培养72 h后,健康对照者CD5+B细胞与CD4+T细胞比例在1∶3时,细胞培养上清液IFN-γ浓度与T细胞单独培养时相比明显下降[(2 427.99±632.62)pg/mL vs.(1 109.25±338.10)pg/mL,P=0.001 2)];而ITP患者CD5+B细胞与CD4+T细胞比例在3∶1时才对IFN-γ的分泌有显著抑制[(3 060.02±493.98)pg/mL vs.(1 769.45±634.73)pg/mL,P=0.000 3]。共培养144 h后得到相似的结果。HD-DXM治疗有效的ITP患者CD5+B细胞与CD4+T细胞比例在1∶1时共培养72 h后,对IFN-γ的分泌有明显抑制[(3 600.02±838.00)pg/mL vs.(1 157.97±383.20)pg/mL,P<0.000 1]。健康对照者及ITP患者CD5+B细胞对CD4+T细胞分泌IL-4均无明显影响。结论原发性ITP患者CD5+B细胞抑制CD4+T细胞分泌Th1型细胞因子IFN-γ的能力明显受损,经HD-DXM治疗后CD5+B细胞抑制IFN-γ分泌的能力可部分恢复。Objective To investigate the regulatory functions of peripheral blood CD5+B cells on the secretion of Th1/Th2 cytokines by CD4+T cells in patients with primary immune thrombocytopenia(ITP)and the immunomodulation alterations of CD5+B cells after treatment with high dose dexamethasone(HDDXM). Methods Peripheral blood mononuclear cells(PBMCs)were isolated from 20 mL peripheral blood of 5 newly-diagnosed primary ITP patients before and after HD-DXM treatment and 5 healthy control subjects using Ficoll density gradient centrifugation.CD5+B cells,CD5-B cells and CD4+T cells were magnetically separated from PBMCs. CD5+B cells or CD5-B cells were cocultured with CD4+T cells for 72 hours or 144 hours at a gradient ratio of 1∶9,1∶5,1∶3,1∶1 and 3∶1.Supernatant IFN-γ(representing Th1 type cytokines) and IL-4(representing Th2 type cytokines) concentration was detected by ELISA. Results In healthy subjects,the concentration of IFN-γ in supernatant of cell culture for 72 hours with the ratio of CD5+B cells to CD4+T cells at 1∶3 was significantly lower than that in T cell culture alone[(2 427.99±632.62)pg/mL vs.(1 109.25±338.10)pg/mL,P=0.001 2].While in ITP patients,CD5+B cells were not observed to decrease IFN-γ secretion until the ratio of CD5+B cells to CD4+T cells increased to 3∶1[(3 060.02±493.98)pg/mL vs.(1 769.45±634.73)pg/mL,P=0.0003]. Similar results were obtained after 144-hours coculture. After HD-DXM therapy,in ITP patients the concentration of IFN-γ after 72-hour coculture was significantly decreased with the ratio of CD5+B cells to CD4+T cells at 1∶1[(3 600.02±838.00)pg/mL vs.(1 157.97±383.20)pg/mL,P<0.000 1].There was no significant effect of CD5+B cells on IL-4 secretion by CD4+T cells both in healthy subjects and in ITP patients. Conclusions The ability of CD5+B cells to suppress the secretion of IFN-γ,one of Th1-type cytokines,is impaired in primary ITP patients. After HD-DXM treatment,the ability of CD5+B cells to inhibit IFN-γ secretion could be partially restored.

关 键 词：免疫性血小板减少症(ITP) CD5+B细胞 TH1/TH2 IFN-γ IL-4 

分 类 号：R554[医药卫生—血液循环系统疾病]