【作者】 张健；

【导师】 徐从高；

【作者基本信息】 山东大学 ， 内科学， 2008， 博士

【摘要】 再生障碍性贫血(简称再障)是一种异质性疾病,多种病因引起骨髓造血功能衰竭,临床表现为全血细胞减少。再障发病机制复杂,涉及造血干细胞损伤、免疫异常、造血微环境改变等因素。目前认为,在大多数病例中,再障是一种获得性的、免疫介导的疾病,伴有T淋巴细胞异常活化造成的造血干细胞破坏。效应性T细胞活化及作为攻击靶点的骨髓造血干/祖细胞损伤的分子机制至今尚未阐明。临床可观察到大多数患者对免疫抑制治疗有所反应,此为免疫系统在再障发病的病理过程中扮演关键角色的有力证明。T淋巴细胞是机体免疫系统中的主要效应细胞,通过对抗原的识别、处理、递呈等作用构成多种细胞同细胞因子间的网络联系,维持机体免疫功能的平衡状态。细胞因子调节的中心是CD4~+T淋巴细胞,即辅助性T淋巴细胞(Th)。Th1/Th2亚群及其相互之间的平衡在免疫应答的调节中起着关键的作用。Th1/Th2分化和转化受多种因素影响,其中局部微环境中细胞因子的调节尤为重要。IL-12是IFN-γ产生的强诱导剂,可促进静息CD4~+T细胞向Th1细胞的分化,IL-23则促进中晚期Th1细胞的进一步分化并作用于记忆T细胞。两者均通过结合于IL-12受体β1亚基(IL-12Rβ1)而诱导Th1细胞的分化以及IFN-γ的产生。已知再障患者血清内源性IL-12显著增高,且体外细胞培养实验中可观察到Tc1的活化及Th1细胞因子的优势。推测抗IL-12受体β1亚基单克隆抗体(anti-IL-12Rβ1mAb)可能通过阻断IL-12信号通路而对再障患者的Th1偏移进行调节。第一部分TIM-3在再生障碍性贫血患者中的表达目的:测定再生障碍性贫血患者血清中细胞因子IFN-γ、IL-18、IL-23、IL-17的浓度,三色流式细胞术检测再障患者及正常对照外周血单个核细胞各亚群Th1特征性表面分子TIM-3表达水平,并通过荧光实时定量RT-PCR检测再障患者外周血单个核细胞及骨髓单个核细胞TIM-3及其他相关因子的表达,探讨TIM-3的表达与再障相关性,为进一步阐明再障的发病机制及TIM-3与再障的关系奠定基础。材料和方法:1.病例选择:再障患者29例,其中重型再障(SAA)16例,非重型再障(NSAA)13例。正常对照31例。(1)抽取空腹外周静脉血10ml,肝素抗凝;(2)抽取清晨空腹外周静脉血5ml,自凝后分离血清;(3)抽取患者髂后上棘骨髓8～10ml或胸骨骨髓约5ml,抽取正常对照骨髓5ml,肝素抗凝。2.密度-梯度法分离外周血单个核细胞及骨髓单个核细胞。3.ELISA检测血清中细胞因子IFN-γ、IL-18、IL-23及IL-17的浓度。4.流式细胞术分析再障患者及正常对照外周血单个核细胞各亚群TIM-3表达水平及CD4~+CD25~+Foxp3~+调节性T细胞的比例。5.荧光实时定量RT-PCR检测再障患者外周血单个核细胞及骨髓单个核细胞IFN-γ、TIM-3的mRNA表达,并进行二者表达的相关性分析。检测外周血单个核细胞galectin-9及Foxp3的mRNA表达。6.统计学处理。结果:1.再障患者血清中细胞因子IFN-γ、IL-18、IL-23、IL-17浓度均显著高于正常对照。依次为:IFN-γ:108.96±35.80 vs.42.41±39.36,pg/ml,p＜0.001;IL-18:485.40±227.08 vs.71.59±73.91,pg/ml,p＜0.001;IL-23:606.88±110.16 vs.213.52±143.10,pg/ml,p＜0.001;IL-17:238.05±173.28 vs.32.38±29.19,pg/ml,p＜0.001。SAA组高于NSAA组(差异界值分别为:p=0.023,p=0.028,p=0.002,p=0.032)。2.正常对照及再障患者不同细胞亚群均可检测到TIM-3表达,正常对照以CD14~+单核细胞、NKT细胞表达较高;而再障患者则以CD4~+T细胞表达较高,其次为CD14~+单核细胞及CD8~+T细胞。再障患者外周血TIM-3~+细胞百分比显著高于正常对照(51.45±10.95 vs.28.11±12.31,%,p＜0.001);TIM-3蛋白的相对表达强度也高于正常对照(68.68±14.76 vs.41.66±15.19,p＜0.001)。3.再障患者外周血CD4~+CD25~+Foxp3~+调节性T细胞的百分比显著低于正常对照(0.69±0.52 vs.2.24±1.56,%,p=0.002),Foxp3的相对表达强度也低于正常对照(51.71±32.12 vs.100.14±60.24,p=0.006)。4.与正常对照相比,再障患者外周血单个核细胞中TIM-3及IFN-γ的mRNA表达值分别增高3.17倍及4.71倍(p＜0.001,p＜0.001)。骨髓单个核细胞中TIM-3及IFN-γ的表达值分别增高4.78倍及7.13倍(p=0.001,p＜0.001)。SAA组高于NSAA组。转录水平IFN-γ的增高与TIM-3的增高在外周血单个核细胞(r=0.806;p＜0.001)及骨髓单个核细胞(r=0.811;p=0.001)均呈正相关。5.与正常对照相比,再障患者外周血单个核细胞中galectin-9 mRNA的表达值增高4倍(p=0.03)。6.再障患者外周血单个核细胞中Foxp3的表达值显著低于正常对照(p=0.001)。结论:1.再障患者血清中细胞因子IFN-γ、IL-18、IL-23、IL-17浓度均显著高于正常。2.再障患者TIM-3在蛋白水平及转录水平均显著高于正常,并与IFN-γ的表达呈正相关,进一步证实再障为Th1优势免疫应答。3.再障患者galectin-9在转录水平显著高于正常,提示TIM-3-galectin-9途径的异常可能参与再障发病。4.再障患者的外周血CD4~+CD25~+Foxp3~+调节性T细胞比例显著降低,且其特征性转录因子Foxp3在蛋白水平及转录水平均显著低于正常,提示再障患者免疫调节能力降低,外周免疫耐受异常。第二部分抗IL-12受体β1亚基单克隆抗体对再生障碍性贫血患者Th1偏移的调节目的:以再障患者来源的骨髓单个核细胞在抗CD3 mAb刺激下培养,观察不同浓度抗IL-12Rβ1 mAb的效应,采用ELISA检测细胞培养上清中IFN-γ的水平;采用荧光实时定量RT-PCR分析培养细胞IFN-γ、T-bet及GATA-3的mRNA表达;并用Western Blot检测培养细胞T-bet、GATA-3、STAT4及磷酸化的STAT4(phospho-STAT4,pSTAT4)的蛋白表达,初步判断抗IL-12Rβ1 mAb在体外纠正再障患者骨髓单个核细胞的Th1偏移状态的可能作用。材料和方法:1.病例选择:再障患者13例,其中SAA7例,NSAA6例。正常对照10例。无菌抽取患者髂后上棘骨髓8～10ml或胸骨骨髓约5ml,抽取正常对照骨髓5ml,肝素抗凝。2.骨髓单个核细胞的培养(1)初次细胞培养:分离来自10例再障患者及5例正常对照的骨髓单个核细胞,不同刺激条件下(未加任何初始刺激组/anti-CD3 mAb 5μg/ml组)培养72h。收集培养上清。(2)不同因素对骨髓单个核细胞的影响:分离来自13例再障患者的骨髓单个核细胞,每例标本均随机分为anti-CD3 mAb(5μg/ml)组、anti-CD3mAb(5μg/ml)+anti-IL-12Rβ1 mAb(0.05μg/ml)组、anti-CD3 mAb(5μg/ml)+anti-IL-12Rβ1 mAb(0.5μg/ml)组及anti-CD3 mAb(5μg/ml)+anti-IL-12Rβ1 mAb(5μg/ml)组。另分离来自10例正常对照的BMMCs,仅给予anti-CD3 mAb(5μg/ml)作为刺激因素。收集细胞及培养上清。3.ELISA检测细胞培养上清中IFN-γ浓度。4.荧光实时定量RT-PCR检测体外培养的再障患者骨髓单个核细胞IFN-γ、T-bet及GATA-3的mRNA表达。5.Western Blot检测体外培养的再障患者骨髓单个核细胞T-bet、GATA-3、STAT4及pSTAT4的蛋白表达。6.统计学处理。结果:1.在没有任何刺激的条件下,多数正常对照骨髓单个核细胞产生IFN-γ量极低(18.64±11.95,pg/ml),而在anti-CD3 mAb刺激后产生IFN-γ显著增加(173.08±67.85,pg/ml,p＜0.001)。再障患者骨髓单个核细胞体外培养不需任何刺激即可产生IFN-γ(127.42±60.16,pg/ml),加入anti-CD3 mAb刺激后IFN-γ分泌显著增加(300.34±99.33,pg/ml,p=0.001)。2.再障患者骨髓单个核细胞培养体系中加入不同浓度anti-IL-12Rβ1 mAb后,细胞培养上清中IFN-γ显著降低(差异界值均为P＜0.001),但仍高于正常对照(差异界值分别为p＜0.001,p=0.014,p=0.034)。3.再障患者骨髓单个核细胞培养体系中加入不同浓度anti-IL-12Rβ1 mAb后,IFN-γ及T-bet在转录水平均有所下调(差异界值分别为IFN-γ:p=0.046,p=0.002,P＜0.001;T-bet:p=0.005,p＜0.001,P＜0.001)。GATA-3在转录水平无显著变化,但T-bet/GATA-3比值降低(差异界值均为p＜0.001)。4.再障患者骨髓单个核细胞培养体系中加入anti-IL-12Rβ1 mAb后,T-bet蛋白表达显著降低(差异界值均为p＜0.001),GATA-3蛋白表达改变不显著,而T-bet/GATA-3比值降低(差异界值均为p＜0.001)。pSTAT4蛋白表达降低(差异界值分别为p=0.048,p＜0.001,p＜0.001),而STAT4蛋白表达变化不显著,pSTAT4/STAT4比值降低(差异界值分别为p=0.046,p＜0.001,p＜0.001)。结论:1.再障患者骨髓单个核细胞处于异常激活状态,不需初始刺激即可分泌IFN-γ。2.Anti-IL-12Rβ1 mAb在转录水平及蛋白水平均可降低体外培养的再障患者骨髓单个核细胞IFN-γ的产生,此作用呈浓度依赖性。3.Anti-IL-12Rβ1 mAb在转录水平及蛋白水平均可降低体外培养的再障患者骨髓单个核细胞Th1转录因子T-bet表达,但对GATA-3的表达影响不显著,并可显著降低T-bet/GATA-3比值,部分纠正Th1偏移,此作用呈浓度依赖性。4.Anti-IL-12Rβ1 mAb对体外培养的再障患者骨髓单个核细胞转录因子STAT4的表达影响不显著,但可显著抑制STAT4磷酸化,降低pSTAT4/STAT4比值。此作用具有浓度依赖性。更多还原

【Abstract】 The pathophysiology of acquired aplastic anemia(AA)can be characterized by a marked reduction in both the quality and quantity of hematopoietic stem cells resulting in pancytopenia.Pathophysiologically,acquired AA seems to be immune-mediated,causing an organ-specific destruction of bone marrow hematopoietic stem cells and progenitor cells through abnormal T cell activation in most patients.The most convicing evidence for the dominant role of the immune system in pathophysiology of AA was lately supported by the efficacy of immunesuppressive treatment in the majority of patients.Nevertheless,understanding of the initiating and perpetuating mechanisms in AA is still incomplete.The imbalance of Th1/Th2 plays an important role in the onset and developmentof AA.Previous studies have also demonstrated endogenous stimulation in AA T cells and supported the Th1/Tc1 autoimmune basis of this complex disease.Aberrant type 1 immune responses have been linked to the pathogenesis of aplastic anemia,and cytokine drives a highly pathogenic T cell population involved in its initiation. Numerous studies have established IL-12 as an important factor for the differentiation ofnaive T cells into IFN-γ-producing Th1 cells,IL-23,a cytokine that shares the p40 subunit with IL-12,has since prompted reevaluation of the role of IL-12/Th1 response in autoimmunity.These cytokines play a pivotal role in the establishment and maintenance of autoimmune diseases and would be new therapeutic targets. PARTⅠEXPRESSION OF TIM-3 AND ITS LIGAND IN APLASTIC ANEMIA PATIENTSObjective:To investigate the Th1 shift in the cytokines and Th1 specific surface molecule in AA patients and to explore the immune mechanism of TIM-3 in aplastic anemia development.Material and methods:1.Bone marrow and peripheral blood samples were collected from 29 AA patients and 31 patients with non-hematopoietic diseases.2.Bone marrow mononuclear cells and peripheral blood mononuclear cells were prepared by ficoll gradient centrifugation technique.3.Concentration of IFN-γ,IL-18,IL-23 and IL-17 in sera from AA patients and controls were determined by ELISA.4.The peripheral blood was incubated with different combination of cell surface antibodies.The analysis of the TIM-3 expression in CD3~+CD4~+(CD4~+ T cells), CD3~+CD8~+(CD8~+ T cells),CD14~+(monocytes),CD19~+(B cells), CD3~-CD16~+CD56~+(NK cells),and CD3~+CD16~+CD56~+(T cells with NK markers) cells were accomplished with flow cytometry.Intracellular staining was performed for analysis of CD4~+CD25~+Foxp3~+ regulatory T cells.The datas were analyzed using the CellQuest software.5.Real-time quantitative RT-PCR was used for relative quantitation of mRNA in bone marrow mononuclear cells and peripheral blood mononuclear cells. Expression of TIM-3,IFN-γ,galectin-9 and Foxp3 transcripts were normalized respectively toβ-actin mRNA.6.The results were expressed in mean±standard deviation(mean±s).Student’s t-test and Spearman’s rank test were performed.P value＜0.05 is considerated as significant.Result:1.The serum concentration of IFN-γ,IL-18,IL-23 and IL-17 all increased in AA patients compared with controls(IFN-γ:108.96±35.80 vs.42.41±39.36,pg/ml, p=0.002;IL-18:485.40±227.08 vs.71.59±73.91,pg/ml,p＜0.001;IL-23: 606.88±110.16 vs.213.52±143.10,pg/ml,p＜0.001;IL-17:238.05±173.28 vs. 32.38±29.19,pg/ml,p＜0.001,respectively).They are both higher in SAA than in NSAA(p=0.001,p=0.028,p=0.002,p=0.032,respectively).2.The major cellular source of TIM-3 in peripheral blood in the controls was CD14~+ monocytes and NKT cells.There was remarkable elevation in TIM-3 protein as well as the frequency of TIM-3~+ cells in peripheral blood mononuclear cells from AA patients compared with controls(68.68±14.76 vs 41.66±15.19,p＜0.001; 51.45±10.95 vs 28.11±12.31,%,p＜0.001,respectively).In AA patients,the majority expressing TIM-3 protein was CD4~+ T cells,CD14~+ monocytes and CD8~+ T cells.3.The frequency of CD4~+CD25~+Foxp3~+T cells in AA patients was much decreased compared with that from the controls(0.69±0.52 vs 2.24±0.16,%,p=0.002).The intracellular Foxp3 protein was also significantly reduced in AA patients (51.71±32.12 vs 100.14±60.24,p=0.006).4.Compared with controls,the expression of TIM-3 and IFN-γmRNA was increased in peripheral blood mononuclear cells from AA patients by 3.17-fold and 4.71-fold respectively(both p＜0.001),and by 4.78-fold and 7.13-fold respectively in bone marrow mononuclear cells of AA patients(p=0.001,p＜0.001, respectively).Significant correlation was determined between the mRNA expression of TIM-3 and IFN-γin both peripheral blood mononuclear cells (r=0.806;p＜0.001)and bone marrow mononuclear cells(r=0.811;p=0.001)from AA patients.5.The expression of galectin-9 transcript increased by 4.0-fold in peripheral blood mononuclear cells from AA patients than controls(p=0.03).6.The expression of Foxp3 transcript significantly decreased in peripheral blood mononuclear cells from AA patients than controls(p=0.001). Conclusion:1.Overproduction of IFN-γ,IL-18,IL-23 and IL-17 was observed in sera from AA patients.2.Realtime RT-PCR results revealed significantly higher mRNA expression of TIM-3 in peripheral blood mononuclear cells and bone marrow mononuclear cells from AA patients compared with controls,which well correlated with IFN-γmRNA.3.Decreased number of regulatory T cell and low expression of Foxp3 were observed in AA patients.4.High expression of galectin-9,the ligand of TIM-3,may be implicated in autoimmune marrow failure.PARTⅡANTI-IL-12Rβ1 mAb REGULATES T-bet AND pSTAT4 EXPRESSION IN BMMCs FROM APLASTIC ANEMIA PATIENTS IN VITROObjective:To investigate the effect of anti-IL-12Rβ1 mAb on Th1 shift in bone marrow mononuclear cells from AA patients in vitro.Material and methods:1.Bone morrow samples were collected from 13 AA patients and 10 patients with non-hematopoietic diseases.2.Bone marrow mononuclear cells were prepared by Ficoll gradient centrifugation technique.(1)Freshly isolated bone marrow mononuclear cells from 10 AA patients and 5 controls were incubated for 72 hours in culture media with anti-CD3 mAb (5μg/ml)or without any stimulation.Supernatant was collected.(2)Freshly isolated bone marrow mononuclear cells from 13 AA patients were cultured by stimulation with anti-CD3 mAb(5μg/ml)in the presence or absence of anti-IL-12Rβ1 mAb for 72 hours.Freshly isolated bone marrow mononuclear cells from 10 controls were cultured by stimulation with anti-CD3 mAb(5μg/ml) for 72 hours.Cell pellets and supernatant were collected. 3.The level of IFN-γin culture supernatant was determined by ELISA.4.Real-time quantitative RT-PCR was used for relative quantitation of mRNA in bone marrow mononuclear cells.Expression of GATA-3,IFN-γand T-bet transcripts were normalized respectively toβ-actin mRNA.5.Western blotting was used for determination of protein in bone marrow mononuclear cells.Expression of GATA-3,pSTAT4,STAT4 and T-bet was normalized respectively toβ-actin protein.6.The results were expressed in mean±standard deviation(mean±s).One-way analysis of variance(ANOVA)or Kruskal-Wallis test were performed.P value＜0.05 is considerated as significant.Result:1.Bone marrow mononuclear cells from controls without any stimulation produced lower level of IFN-γin supernatants(18.64±11.95,pg/ml).IFN-γproduction was elevated by 9.29-fold by stimulation with anti-CD3 mAb(p＜0.001).Bone marrow mononuclear cells from AA patients without any stimulation produced increased levels of IFN-γcompared with controls(127.42±60.16,pg/ml,p＜0.001).IFN-γproduction was elevated by 2.36-fold by stimulation with anti-CD3 mAb (p=0.001).2.Anti-IL-12Rβ1 mAb significantly decreased production of IFN-γin culture supernatant in a dose-dependent manner(all p＜0.001).3.Anti-IL-12Rβ1 mAb significantly decreased expression of IFN-γand T-bet at transcript level in anti-CD3 mAb-stimulated bone marrow mononuclear cells from AA patients in a dose-dependent manner(p=0.046,p=0.002,p＜0.001 for IFN-γ; p=0.002,p＜0.001,p＜0.001 for T-bet,respectively).4.Anti-IL-12Rβ1 mAb significantly decreased expression of T-bet and pSTAT4 at protein level in anti-CD3 mAb-stimulated bone marrow mononuclear cells from AA patients in a dose-dependent manner(all p＜0.001 for T-bet;p=0.048,p＜0.001, p＜0.001 for pSTAT4,respectively). Conclusion:1.There is aberrant activation of Th1 cell in AA resulting in overproduction of IFN-γwithout any prior stimulation.2.Anti-IL-12Rβ1 mAb may significantly decrease expression of IFN-γat both transcript and protein levels in a dose-dependent manner in anti-CD3 mAb-stimulated bone marrow mononuclear cells from AA patients in vitro.3.Anti-IL-12Rβ1 mAb may significantly decrease expression of T-bet at both transcript and protein levels in a dose-dependent manner in anti-CD3 mAb-stimulated bone marrow mononuclear cells from AA patients in vitro.4.Anti-IL-12Rβ1 mAb may significantly decrease pSTAT4 at protein level in a dose-dependent manner in anti-CD3 mAb-stimulated bone marrow mononuclear cells from AA patients in vitro.更多还原