【作者】 胡晓静；

【导师】 徐从高；

【作者基本信息】 山东大学 ， 内科学， 2009， 博士

【摘要】 再生障碍性贫血（简称再障）是一种骨髓衰竭性疾病,以骨髓造血功能低下和全血细胞减少为特征。近年来认为,再障的主要发病机制是T细胞介导的骨髓特异性的自身免疫反应。根据T细胞表面跨膜蛋白酪氨酸磷酸酶CD45RA和趋化因子受体CCR7的表达与否,T细胞可分为初始T细胞、中心记忆性T细胞(T_CM细胞)和效应记忆性T细胞(T_EM细胞)亚群。研究发现再障患者中存在T细胞优势克隆群,呈寡克隆扩增的T细胞具有自身反应性,显示出记忆/效应细胞表型,提示记忆性T细胞亚群可能在再障的发病过程中发挥重要作用。T细胞表面钾离子通道与T细胞的活化有着极为重要的关系。人类T细胞表面两种钾离子通道,电压门控钾通道Kv1.3和钙依赖性钾通道KCa3.1可通过对膜电位和Ca²⁺信号通路的调控,调节T细胞的活化和功能。不同T细胞亚群活化时,钾离子通道的表达有所不同。T_EM细胞活化时高表达Kv1.3通道,而低表达KCa3.1通道,Kv1.3通道是T_EM细胞活化的主要离子通道。而在初始T细胞和T_CM活化时,KCa3.1通道表达增加,发挥主要功能,Kv1.3通道无明显增加。阻断Kv1.3通道,可以有效抑制T_EM细胞的增殖及效应功能。本研究检测了再障初始和记忆性T细胞亚群的变化及T细胞活化时Kv1.3通道的表达,分析Kv1.3通道在再障T细胞及初始、记忆性T细胞亚群活化中的作用,探讨Kv1.3通道作为再障免疫治疗靶点的可能性。第一部分再生障碍性贫血患者初始和记忆性T细胞亚群数量及功能的研究目的:研究再障患者骨髓及外周血CD4⁺及CD8⁺T细胞中初始T细胞、T_CM细胞和T_EM细胞的数量及分泌细胞因子IFN-γ的功能。方法:1.再障外周血及骨髓中初始和记忆性T细胞亚群数量的检测:抽取再障患者和正常对照外周血及骨髓液进行单个核细胞的分离,流式细胞术检测外周血单个核细胞（PBMCs）及骨髓单个核细胞（BMMNCs）的CD4⁺及CD8⁺T细胞中初始T细胞（CD45RA⁺CCR7⁺）、T_CM细胞（CD45RA^-CCR7⁺）和T_EM细胞(CD45RA^-CCR7^-,CD8⁺T细胞还包括终末分化的CD45RA⁺CCR7^-T_EM细胞)的比例。2.再障外周血及骨髓中初始和记忆性T细胞亚群功能的检测:培养再障患者和正常对照外周血及骨髓单个核细胞,加入佛波酯PMA、离子霉素刺激T细胞活化,莫能霉素阻断细胞因子向胞外释放。培养5h后,流式细胞术检测CD4⁺及CD8⁺T细胞中CCR7⁺初始T细胞/T_CM细胞和CCR7^-T_EM细胞IFN-γ的表达情况。结果:1.再障患者与正常对照的初始和记忆性T细胞亚群的比较:在PBMCs及BMMNCs的CD4⁺T细胞中,再障患者初始T细胞的比例明显低于正常对照（p＜0.001）,再障患者T_EM细胞的比例明显高于正常对照（p＜0.001）,T_CM细胞比例在再障患者及正常对照之间无明显差异。在PBMCs及BMMNCs的CD8⁺T细胞中,再障患者初始T细胞的比例明显低于正常对照（p＜0.001）,而T_EM细胞及终末T_EM细胞的比例均明显高于正常对照（p＜0.001）,T_CM细胞比例在再障患者及正常对照之间无明显差异。2.外周血与骨髓中初始和记忆性T细胞亚群的比较:再障患者的CD4⁺初始和记忆性T细胞亚群,在外周血与骨髓之间未见明显差异;正常对照的CD4⁺T细胞亚群,在外周血与骨髓之间也未见明显差异。而再障患者骨髓中的CD8⁺终末T_EM细胞的比例较外周血中明显增加（p=0.002）,CD8⁺初始T细胞的比例较外周血中减少（p＜0.001）;正常对照的CD8⁺初始和记忆性T细胞亚群,在外周血与骨髓之间未见明显差异。3.重型再障（SAA）患者与非重型再障（NSAA）患者的初始和记忆性T细胞亚群的比较:PBMCs及BMMNCs中的CD4⁺T细胞亚群,SAA组与NSAA组间未见明显差异;PBMCs及BMMNCs中的CD8⁺T细胞亚群,两组间也未见明显差异。4.初始和记忆性T细胞亚群IFN-γ的表达水平:在再障患者和正常对照的PBMCs及BMMNCs的CD4⁺T细胞中,IFN-γ⁺CCR7^-T_EM细胞比例明显高于IFN-γ⁺CCR7⁺初始T细胞/T_CM细胞（p＜0.001）;同样的,CD8⁺T细胞中,IFN-γ⁺CCR7^-T_EM细胞比例明显高于IFN-γ⁺CCR7⁺初始T细胞/T_CM细胞（p＜0.001）。5.再障患者与正常对照的初始和记忆性T细胞亚群IFN-γ表达水平的比较:再障患者PBMCs及BMMNCs的CD4⁺和CD8⁺T细胞中,IFN-γ⁺CCR7^-T_EM细胞比例均明显高于正常对照（p＜0.001）;而在PBMCs及BMMNCs的CD4⁺和CD8⁺T细胞中,再障患者及正常对照的IFN-γ⁺CCR7⁺初始T细胞/T_CM细胞比例未见明显差异。在再障患者PBMCs及BMMNCs的CD4⁺CCR7^-T_EM细胞中,表达IFN-γ的细胞比例较正常对照无明显增高;而在再障患者PBMCs及BMMNCs的CD8⁺CCR7^-T_EM细胞中,表达IFN-γ的细胞比例较正常对照明显增高（p＜0.001）。结论:再障外周血及骨髓CD4⁺和CD8⁺T细胞中T_EM细胞数量明显增高,骨髓中CD8⁺终末T_EM细胞又较外周血中明显增高;再障的T_EM细胞,较自身初始T细胞、T_CM细胞及正常对照的T_EM细胞,可表达大量造血负调控因子IFN-γ,提示T_EM细胞是再障中主要的效应细胞,可能参与再障的发病过程。第二部分电压门控钾通道Kv1.3在再生障碍性贫血患者T细胞中的表达目的:研究再障患者骨髓活化T细胞中Kv1.3通道的表达情况。方法:1.骨髓T细胞的活化:抽取再障患者和正常对照的骨髓液,进行T细胞的分离,以抗CD3单抗刺激T细胞活化,培养72h。2.荧光实时定量RT-PCR检测再障活化T细胞中Kv1.3通道mRNA表达水平。3.Western blot检测再障活化T细胞中Kv1.3通道蛋白表达水平。4.荧光显微镜检测再障活化T细胞膜表面Kv1.3通道及CCR7的表达情况。5.流式细胞术检测再障活化CD4⁺及CD8⁺T细胞中初始T细胞、T_CM细胞和T_EM细胞的比例。结果:1.再障患者骨髓T细胞活化后Kv1.3通道mRNA的表达与正常对照相比明显增高（p=0.001）。2.再障患者骨髓T细胞活化后Kv1.3通道蛋白的表达与正常对照相比明显增高（p=0.001）。3.荧光显微镜下,再障患者骨髓活化T细胞主要为低表达CCR7的CCR7^-T_EM细胞,这些细胞Kv1.3通道呈现高表达;正常对照的T细胞主要为高表达CCR7的CCR7⁺初始T细胞/T_CM细胞,而这些细胞表面低表达Kv1.3通道。4.流式细胞术结果显示,在骨髓活化的CD4⁺T细胞中,再障患者T_EM细胞的比例明显高于正常对照（p＜0.001）,再障患者初始T细胞的比例明显低于正常对照（p＜0.001）,T_CM细胞比例在再障患者及正常对照之间无明显差异。在CD8⁺T细胞中,再障患者T_EM细胞及终末T_EM细胞的比例均明显高于正常对照（分别为p＜0.001,p=0.001）,初始T细胞的比例明显低于正常对照（p＜0.001）,T_CM细胞比例在再障患者及正常对照之间无明显差异。T_EM细胞是再障患者骨髓活化T细胞中的主要细胞群体。结论:再障患者活化的骨髓T细胞中Kv1.3通道在mRNA及蛋白水平的表达较正常明显增高,这与再障T细胞中T_EM细胞的数量增加及T_EM细胞活化时高表达Kv1.3通道有关。第三部分阻断Kv1.3通道对再生障碍性贫血患者T细胞及T细胞亚群的影响目的:研究阻断Kv1.3通道对再障患者骨髓T细胞及T细胞亚群增殖及功能的影响。方法:1.骨髓T细胞的培养:抽取再障患者骨髓液,进行T细胞的分离,以抗CD3单抗刺激T细胞活化的同时,加入或不加入Kv1.3通道特异性阻断剂ShK,培养72h。2.阻断Kv1.3通道对再障骨髓T细胞增殖及功能的影响的检测:CCK-8法检测不同处理组再障患者T细胞增殖的情况,ELISA法检测不同处理组培养上清中IFN-γ及IL-4的浓度。3.CFSE荧光标记法检测阻断Kv1.3通道对再障骨髓T细胞亚群增殖的影响:再障患者骨髓T细胞刺激前标记CFSE荧光染料,培养72h后通过流式细胞术检测CFSE的变化,分析不同处理组CD4⁺和CD8⁺T细胞中CCR7⁺初始T细胞/T_CM细胞、CCR7^-T_EM细胞增殖的变化。4.流式细胞术检测阻断Kv1.3通道对再障骨髓T细胞亚群功能的影响:分析不同处理组CD4⁺和CD8⁺T细胞中CCR7⁺初始T细胞/T_CM细胞、CCR7^-T_EM细胞表达细胞因子IFN-γ及IL-4水平的变化。结果:1.Kv1.3通道阻断剂ShK可明显抑制再障患者骨髓T细胞的增殖（p＜0.001）。2.阻断Kv1.3通道对再障骨髓T细胞分泌细胞因子水平的影响:ShK加入培养体系后,再障患者培养上清中IFN-γ水平显著降低（p＜0.001）,IL-4水平未见明显改变。3.阻断Kv1.3通道对再障骨髓T细胞亚群增殖的影响:CFSE^low细胞代表增殖的细胞,在ShK加入培养体系后,CD4⁺CCR7^-T_EM细胞及CD8⁺CCR7^-T_EM细胞中CFSE^low细胞比例均显著降低（p＜0.001）,而CD4⁺CCR7⁺初始T细胞/T_CM细胞及CD8⁺CCR7⁺初始T细胞/T_CM细胞中CFSE^low细胞比例未见明显降低。4.阻断Kv1.3通道对再障骨髓T细胞亚群表达细胞因子水平的影响:CD4⁺及CD8⁺T细胞中,分泌IFN-γ和IL-4的细胞主要为CCR7^-T_EM细胞。ShK加入培养体系后,CD4⁺CCR7^-T_EM细胞及CD8⁺CCR7^-T_EM细胞中表达IFN-γ的细胞比例显著降低（分别为p=0.002,p＜0.001）,而CD4⁺CCR7⁺初始T细胞/T_CM细胞及CD8⁺CCR7⁺初始T细胞/T_CM细胞中表达IFN-γ的细胞比例未见明显降低;CD4⁺和CD8⁺CCR7⁺初始T细胞/T_CM细胞及CD4⁺和CD8⁺CCR7^-T_EM细胞中表达IL-4的细胞比例均未见明显降低。结论:特异性阻断Kv1.3通道,可抑制再障T细胞的增殖及功能,其中主要抑制T_EM细胞亚群的增殖及功能,对初始T细胞及T_CM细胞影响不大;Kv1.3通道在再障T_EM细胞活化过程中发挥重要作用;Kv1.3通道可能成为再障免疫治疗的新靶点。更多还原

【Abstract】 Aplastic anemia（AA） is a bone marrow failure disorder characterized by peripheral blood pancytopenia and bone marrow hypoplasia.Immune-mediated suppression of hematopoiesis is considered as the pivotal mechanism responsible for bone marrow failure in this disease.Based on the ability to express chemokine receptor CCR7 and the phosphatase CD45RA,T cells can be divided into na（l|¨）ve T cells,central memory T cells(T_CM cells),and effector memory T cells(T_EM cells).Oligoclonal T cells are described to expand in AA and detected to show mature memory/effector phenotypes, implicating memory T cells may participate in pathophysiologic process of AA.Potassium channels play an important role in T cell activation.Two lymphocyte potassium channels,the voltage-gated potassium channel Kv1.3 and the calcium-dependent potassium channel KCa3.1 could regulate the T cell activation by modulating membrane potential and Ca²⁺ signaling in T cells.In parallel with the differentiation from na（l|¨）ve into T_EM cells,the expression pattern of the lymphocyte potassium channels changes drastically.Kv1.3 channels are up-regulated in activated T_EM cells and become the dominant functional potassium channels.In contrast,in na（l|¨）ve T cells and T_CM cells,the number of Kv1.3 channels increases only modestly after activation,while KCa3.1 channels are up-regulated as the dominant functional potassium channels.Blocking Kv1.3 channels could inhibit the proliferation and function Of T_EM cells.In this study,we detected the frequency and function of na（l|¨）ve T cells,T_CM cells and T_EM cells in AA,investigated the expression of Kv1.3 channels in activated T cells from AA,evaluated the role of Kv1.3 channels in the activation of T cells and T-cell subsets from AA,and discussed the possibility of Kv1.3 channel as a therapeutic target for AA.PartⅠFrequency and function of na（l|¨）ve and memory T-cell subsets in aplastie anemiaObjective:To detect the frequency and function of na（l|¨）ve T cells,T_CM cells and T_EM cells in CD4⁺ and CD8⁺ T cells from bone marrow and peripheral blood of patients with AA.Methods:1.Peripheral blood mononuclear cells（PBMCs） and bone marrow mononuclear cells （BMMNCs） were separated by Ficoll-Hypaque density gradient centrifugation. The percentages of na（l|¨）ve T cells（CD45RA⁺CCR7⁺）,T_CM cells（CD45RA^-CCR7⁺） and T_EM cells(CD45RA^-CCR7^-,CD8⁺ T cells also contain terminally differentiated CD45RA⁺CCR7^- T_EM cells) in CD4⁺ and CD8⁺ T cells in PBMCs and BMMNCs of patients with AA and controls were analyzed by flow cytometry.2.Isolated PBMCs and BMMNCs of patients with AA and controls were simulated with PMA and ionomycin in the presence of monesin for 5h.The expression of intracellular cytokine IFN-γin CD4⁺/CD8⁺ CCR7⁺ na（l|¨）ve/T_CM cells and CCR7^-T_EM cells were analyzed by flow cytometry.Results:1.Comparison of percentages of T-cell subsets in PBMCs and BMMNCs between patients with AA and controls:In the CD4⁺ T-cell population,significantly decreased percentages of na（l|¨）ve T cells were observed in PBMCs and BMMNCs of patients with AA compared with controls（p＜0.001）.In contrast,the percentages of T_EM cells in PBMCs and BMMNCs were higher in patients with AA than in controls（p＜0.001）.As far as CD8⁺ T-cell population was concerned, patients with AA still had decreased percentages of na（l|¨）ve T cells in PBMCs and BMMNCs compared with controls（p＜0.001）.Meanwhile,both T_EM cells and terminal T_EM cells were increased in PBMCs and BMMNCs of patients with AA compared with controls（p＜0.001）.There was no significant difference between patients with AA and controls in the percentages of CD4⁺ and CD8⁺ T_CM cells from PBMCs or BMMNCs.2.Comparison of percentages of T-cell subsets between PBMCs and BMMNCs: There were no significant differences of T-cell subsets in CD4⁺ T cells between PBMCs and BMMNCs in both patients with AA and controls.Concerning CD8⁺ T-cell population,significantly increased terminal T_EM cells and decreased na（l|）ve T cells were observed in BMMNCs compared with PBMCs in patients with AA （p=0.002,p＜0.001,respectively）,but no difference was detected in controls.3.Comparison of percentages of T-cell subsets in BMMNCs and PBMCs between SAA and NSAA patients:In CD4⁺ and CD8⁺ T cell population of PBMCs and BMMNCs,no significant difference in percentages of na（l|¨）ve and memory T-cell subsets was detected between SAA and NSAA.4.The percentages of IFN-γ⁺ na（l|¨）ve and memory T-cell subsets in CD4⁺ and CD8⁺ T cells:In CD4⁺ and CD8⁺ T cells,the percentages of IFN-γ⁺CCR7^- T_EM cells from both PBMCs and BMMNCs were significantly higher than IFN-γ⁺CCR7⁺ na（l|¨）ve/T_CM cells in patients with AA as well as in controls（p＜0.001）.5.Comparison of the expression of intracellular cytokine IFN-γin T-cell subsets between patients with AA and controls:Among total CD4⁺ or CD8⁺ T cells,the percentages of IFN-γ⁺CCR7^- T_EM cells were significantly higher from PBMCs and BMMNCs in patients with AA than that in controls（p＜0.001）.The percentages of IFN-γproducers in CD4⁺CCR7^- T_EM cells from PBMCs or BMMNCs did not differ between patients with AA and controls;whereas the production of IFN-γin CD8⁺CCD7^- T_EM cells from both PBMCs and BMMNCs was significantly higher in patients with AA than controls（p＜0.001）.Conclusions:CD4⁺ T_EM cells and CD8⁺ T_EM cells are increased in peripheral blood and bone marrow of AA,and CD8⁺ terminal T_EM cells are preferentially increased in bone marrow of AA.T_EM cells in AA have increased effector capacity compared with na（l|¨）ve/T_CM cells in AA and T_EM cells in controls.Increased T_EM cells,particularly in marrow of AA,may react as effector cells and participate in immune-mediated suppression of hematopoiesis in AA. PartⅡExpression of the voltage-gated potassium channel Kv1.3 in T cells of aplastic anemiaObjective:To investigate the expression of Kv1.3 channels in activated T cells from bone marrow of patients with AA.Methods:1.T cells were isolated from bone marrow of patients with AA and controls,and then stimulated for 72h with anti-CD3 antibody for T cell activation.2.Kv1.3 mRNA was detected in activated T cells of AA using real-time RT-PCR.3.Kv1.3 protein was detected in activated T cells of AA using western blot.4.The expression of Kv1.3 and CCR7 on activated T cells was detected by immunofluorescence microscopy.5.The percentages of na（l|¨）ve T cells,T_CM cells and T_EM cells in activated CD4⁺ and CD8⁺ T cells were analyzed by flow cytometry.Results:1.The expression of Kv1.3 mRNA in activated T cells was higher in patients with AA than in controls（p=0.001）.2.The expresstion of Kv1.3 protein in activated T cells was higher in patients with AA than in controls（p=0.001）.3.Immunofluorescence microscopy analysis revealed that activated T cells of patients with AA were mainly CCR7^- T_EM cells,having Kv1.3^high phenotype.In contrast,activated T cells of controls were mainly CCR7⁺ na（l|¨）ve/T_CM cells,and did not exhibit conspicuous membrane Kv1.3 staining.4.The percentages of T_EM cells in activated CD4⁺ T cells were higher in patients with AA than in controls（p＜0.001）.The percentages of both T_EM cells and terminal T_EM cells in activated CD8⁺ T cells were higher in patients with AA than in controls（p＜0.001,p=0.001,respectively）.Decreased percentages of na（l|¨）ve T cells were observed in activated CD4⁺ and CD8⁺ T cells of patients with AA compared with controls（p＜0.001）.There was no significant difference between patients with AA and controls in the percentages of CD4⁺ and CD8⁺ T_CM cells. Conclusions:Kv1.3 expression is elevated at mRNA and protein levels in activated T cells from bone marrow of patients with AA.Activated T_EM cells in AA have Kv1.3^high phenotype.Higher Kv1.3 expression in the increased T_EM cells of AA causes the elevated Kv1.3 expression in activated T cells.PartⅢEffects of blocking Kv1.3 channels on proliferation and function of T cells and T-cell subsets in aplastic anemiaObjective:To evaluate the effects of blocking Kv1.3 channels on proliferation and function of T cells and T-cell subsets from bone marrow of patients with AA.Methods:1.T cells were isolated from bone marrow of patients with AA,and then stimulated by anti-CD3 antibody with ShK or without ShK for 72h.2.The effects of blocking Kv1.3 channels with ShK on T cell proliferation were measured by CCK-8 assay.Concentration of IFN-γand IL-4 in supernatant was measured by ELISA.3.CFSE staining was used to investigate the effects of blocking Kv1.3 channels with ShK on proliferation of different T-cell subsets.T cells were labelled with fluorochrome CFSE.After stimulated for 72h,the proliferation of CD4⁺/CD8⁺ CCR7⁺ na（l|¨）ve/T_CM cells and CCR7^- T_EM cells was analyzed by analyzing the dilution of CFSE signal in the FL1 channel by flow cytometry.4.Intracellular cytokine production staining was used to detect the effects of blocking Kv1.3 channels with ShK on function of different T-cell subsets.The expression of intracellular cytokine IFN-γ,and IL-4 in CD4⁺/CD8⁺ CCR7⁺ na（l|¨）ve/T_CM cells and CCR7^- T_EM cells was analyzed by flow cytometry.Results:1.As shown by CCK-8 analysis,the proliferation of T cells treated with ShK was significantly decreased in patients with AA compared with T cells stimulated by anti-CD3 without ShK（p＜0.001）.2.The IFN-γproduction of T cells from patients with AA reduced significantly in the presence of ShK（p＜0.001）.The suppression of IL-4 production induced by ShK was unremarkable in AA.3.CFSE^low cells represented the proliferating cells.In the presence of ShK,the percentages of CFSE^low cells reduced significantly in CD4⁺CCR7^- T_EM cells and CD8⁺CCR7^- T_EM cells of AA（p＜0.0001）.But ShK exerted unremarkable inhibitory effects on the percentages of CFSE^low cells in CD4⁺CCR7⁺ na（l|¨）ve/T_CM cells and CD8⁺CCR7⁺ na（l|¨）ve/T_CM cells.4.IFN-γand IL-4-producing CD4⁺/CD8⁺ T cells were mainly CCR7^- T_EM cells. When T cells were stimulated by anti-CD3 in the presence of ShK,the percentages of IFN-γ⁺ cells in CD4⁺CCR7^- T_EM cells and CD8⁺CCR7^- T_EM was decreased significantly（p=0.002,p＜0.001,respectively）.But ShK exhibited unremarkable inhibitory effects upon intercellular IFN-γexpression in CD4⁺CCR7⁺ na（l|¨）ve/T_CM cells and CD8⁺CCR7⁺ na（l|¨）ve/T_CM cells.Little intracellular IL-4 expressed in T cells from AA and the inhibition of ShK was unremarkable.Conclusions:Blocking Kv1.3 channels could inhibit the proliferation and function of T cells in bone marrow of AA.And inhibitory effects are mainly on the proliferation and type-1 response of T_EM cells.Kv1.3 channels are the dominant functional potassium channels of the activated T_EM cells in AA.Therapy targeting to Kv1.3 channels in T_EM cells may improve current immunosuppressive therapies for AA without influencing the whole immune system.更多还原