【作者】 王迎雪；

【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（再障）是由多种病因引起的骨髓造血功能衰竭症,主要表现为骨髓造血功能低下、全血细胞减少。从发病学上再障可分为遗传性和获得性。获得性再障更为常见,其中大部分获得性再障无明确诱因可寻,称为原发性获得性再障。本文所研究的是指原发性获得性再障,以下均简称再障。再障的发病机制复杂,涉及造血干细胞损伤,免疫异常,造血微环境改变等因素。目前多数研究认为,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,免疫抑制治疗则是目前临床治疗再障的主要手段。有关诱发再障患者免疫反应的干细胞起始抗原的研究较少,对其与再障发病相关性的研究还在进行中,更多的研究主要是集中在T细胞免疫方面。许多研究证实,再障患者存在细胞免疫异常,包括T细胞异常活化,T细胞亚群分布及表型表达异常,CD4⁺/CD8⁺细胞比例倒置,Th1/Th2细胞比值升高,T细胞分泌的造血负调控因子TNF-α、IFN-γ、IL-2浓度升高,结果导致再障骨髓造血干/祖细胞数量减少和增殖能力下降。自然杀伤T（NKT）细胞是一类具有NK细胞受体和T细胞受体且显示NK细胞和T细胞两方面性质的T细胞亚群,表达保守的限制性的TCR谱,人NKT细胞由恒定的Vα24 Jα18TCRα链和Vβ11TCRβ链组成,在调节自身免疫中起了重要作用。NKT细胞很重要的功能特征是它们在体外受到刺激后活化,可以迅速分泌大量的细胞因子,包括Th1型细胞因子（IFN-γ,TNF-α等）或Th2型细胞因子（IL-4,IL-10等）,影响免疫反应的类型,使免疫反应向Th1型或Th2型转换。诱导NKT细胞产生Th1或Th2型细胞因子依赖于刺激的类型、强度、时间和免疫微环境,在不同刺激因子的作用下,其分泌的细胞因子也不同。Th1和Th2细胞通过分泌的细胞因子进行发生、分化的相互调节。因此,在不同的情况下,NKT细胞可能通过释放不同的细胞因子来调节Th1/Th2的分化,是一种重要的免疫调节细胞。作为可影响Th1/Th2分化的免疫调节细胞,NKT细胞在再障细胞免疫异常的发病机制中所处的地位和作用,目前国内外对此研究甚少,尚无这方面的报导。本研究第一部分检测再障患者骨髓中NKT细胞的数量以及不同条件下体外活化后的细胞因子分泌功能状态,与正常入骨髓中NKT细胞的数量及功能状态进行比较;第二部分建立再障患者骨髓造血细胞液态扩增培养体系及半固体集落培养体系,研究NKT细胞经α-Galcer活化后对液态培养体系中Th1/Th2型细胞因子水平的影响,以及对集落培养体系中CFU-GM形成的影响。从而探讨再障患者骨髓中NK7细胞的病理状态、在再障发病免疫机制中所起的作用,探寻α-Galcer作用下NKT细胞的免疫调节对恢复再障Th1/Th2平衡的作用,寻求再障免疫调节治疗的新方向。第一部分再障患者骨髓中TCRVα24⁺Vβ11⁺ NKT细胞的数量以及体外活化后的功能状态研究目的:检测再障患者骨髓中TCRVα24⁺Vβ11⁺NKT细胞的数量以及体外活化后的功能状态,与正常入骨髓中NKT细胞的数量及功能状态进行比较,初步判断再障患者骨髓中NKT细胞的变化或病理状态,并研究NKT细胞在不同条件刺激下扩增活化后,Th1型、Th2型细胞因子表达的不同状况,从而探寻NKT细胞在再障免疫异常中的作用以及在免疫调节治疗中潜在意义的实验室依据。材料和方法:1.研究对象再障患者20例,其中重型再障（SAA）8例,非重型再障（NSAA）12例。正常对照20例。2.骨髓单个核细胞（BMMNCs）的制备无菌抽取再障患者髂后上棘骨髓8-10ml或胸骨骨髓5ml,抽取正常对照骨髓5ml,肝素抗凝。密度-梯度法分离BMMNCs。3.再障患者与正常对照BMMNCs中NKT细胞的扩增活化将制备的BMMNCs置于含10%灭活胎牛血清的RPMI1640培养液,调整细胞浓度为1×10⁶/ml,于24孔培养板中培养,1ml/孔,分为2组,加入α-Galcer及细胞因子不同的组合,组①:α-Galcer+rhIL-2;组②:α-Galcer+rhIL-2+rhG-CSF,置于含5%CO₂、饱和湿度的37℃培养箱中培养7天。计数扩增后MNCs数目。4.NKT细胞数量及扩增能力检测流式细胞术检测扩增前及扩增后MNCs中TCRVα24⁺Vβ11⁺ NKT细胞的比率,计算NKT细胞数目,并计算NKT细胞的扩增倍数。5.NKT细胞活化后细胞内因子检测BMMNCs扩增活化7天后,在培养细胞中加入甲醇溶解的莫能霉素（2ug/ml）,培养4小时后按照细胞固定破膜剂使用说明处理细胞,流式细胞术测定TCRVα24⁺Vβ11⁺NKT细胞中胞内IFN-γ、IL-4阳性细胞的比率。6.统计学处理结果:1.再障患者与正常对照骨髓中NKT细胞的含量再障患者与正常对照BMMNCs中TCRVα24⁺+Vβ11⁺NKT细胞的细胞比率分别为（0.18±0.09）%、（0.26±0.13）%（P＜0.05）。2.再障患者与正常对照骨髓中NKT细胞的扩增能力在组①α-Galcer+rhIL-2培养体系中,再障患者BMMNCs中NKT细胞的扩增倍数较正常对照显著降低（80.45±43.84 vs 120.44±65.32,P＜0.05）;在组②α-Galcer+rhIL-2+rhG-CSF培养体系中,再障患者BMMNCs中NKT细胞的扩增倍数仍较正常对照显著降低（66.96±32.59 vs 99.03±58.58,P＜0.05）。在组①α-Galcer+rhIL-2培养体系中加入rhG-CSF后,再障患者及正常对照骨髓中NKT细胞的扩增能力均显著降低（P＜0.05）。3.再障患者与正常对照骨髓中NKT细胞活化后胞内细胞因子的表达BMMNCs在组①α-Galcer+rhIL-2培养体系中扩增活化7天后,再障患者的NKT细胞表达IFN-γ显著高于正常对照（63.55%±15.27%vs 51.48%±13.36%,P＜0.05）,而两者间IL-4的表达无显著差异（27.24%±10.86%vs 31.98%±10.44%,P＞0.05）。BMMNCs在组②α-Galcer+rhIL-2+rhG-CSF的培养体系中扩增活化7天后,再障患者的NKT细胞表达IFN-γ仍显著高于正常对照（36.98%±7.70%vs27.17%±10.44%,P＜0.05）,而IL-4的表达则显著低于正常对照（55.64%±12.76%vs68.70%±15.82%,P＜0.05）。在组①α-Galcer+rhIL-2培养体系中加入rhG-CSF,再障患者与正常对照BMMNCs中NKT细胞扩增活化后,细胞内IFN-α的表达均较组①显著降低（P＜0.001）,而IL-4的表达则均较组①显著升高（P＜0.001）。结论:1.再障患者骨髓中NKT细胞较正常对照显著减少,经α-Galcer刺激后,其扩增能力较正常对照显著降低,并且较正常对照高表达Th1型细胞因子IFN-γ。2.在rhG-CSF作用下,再障患者及正常对照BMMNCs中NKT细胞经α-Oalcer刺激后的扩增能力下降,并且NKT2型细胞优势扩增,细胞因子分泌功能向Th2型细胞因子偏移,IFN-γ表达降低,IL-4表达升高。第二部分NKT细胞经α-Ga1cer刺激活化后对再障患者骨髓造血恢复影响的研究目的:建立再障患者及正常对照骨髓造血细胞液态扩增培养体系及半固体集落培养体系,研究NKT细胞经α-Galcer活化后对液态培养体系中Th1、Th2型细胞因子水平的影响,以及对集落培养体系中CFU-GM形成的影响,初步探讨NKT细胞应α-Galcer刺激活化后对再障骨髓造血功能恢复的作用。材料和方法:1.研究对象再障患者15例,其中重型再障（SAA）7例,非重型再障（NSAA）8例。正常对照15例。2.骨髓单个核细胞（BMMNCs）的制备无菌抽取患者髂后上棘骨髓8～10ml或胸骨骨髓5ml,抽取正常对照骨髓5ml,肝素抗凝。密度-梯度法分离BMMNCs。3.骨髓造血细胞体外液态培养扩增将制备的BMMNCs置于含10%灭活胎牛血清的IMDM培养液,调整细胞浓度为2×10⁵/ml,于24孔培养板中培养,1ml/孔,分为2组,每组三复孔。组（1）加入rhG-CSF+rhSCF,组（2）加入α-Galcer+rhG-CSF+rhSCF。置于含5%CO₂、饱和湿度的37℃培养箱中培养。72小时后离心收集各组培养上清。4.细胞因子检测应用ELISA法测定各组培养上清中IFN-γ、IL-4的含量。5.骨髓造血细胞CFU-GM培养将制备的BMMNCs置于含1.5%甲基纤维素、30%灭活胎牛血清的IMDM半固体培养基,调整细胞浓度为1×10⁵/ml,于24孔培养板中培养,1ml/孔,分为2组,每组三复孔。组（1）加入rhG-CSF+rhSCF,组（2）加入α-Galcer+rhG-CSF+rhSCF。置于含5%CO₂、饱和湿度的37℃培养箱中培养。7～10天时观察计数形成的CFU-GM。6.统计学处理结果:1.再障患者与正常对照骨髓造血细胞液态扩增培养后上清中细胞因子的水平在组（1）rhG-CSF+rhSCF及组（2）α-Galcer+rhG-CSF+rhSCF两种液态培养体系中,再障患者BMMNCs扩增培养后上清中IFN-γ水平分别为（1739.34±604.90）ng/L、（981.72±266.73）ng/L（P＜0.001）,而IL-4水平分别为（105.19±31.31）ng/L、（305.73±51.96）ng/L（P＜0.001）。正常对照BMMNCs在组（1）及组（2）两组体系中扩增培养后,上清中IFN-γ水平分别为（531.58±120.63）ng/L、（405.04±109.27）ng/L（P＜0.001）,而IL-4水平分别为（53.58±27.54）ng/L、（246.36±77.01）ng/L（P＜0.001）。同时上述数据表明,再障患者BMMNCs在两个培养体系中扩增后,与同样条件下的正常对照相比,上清中IFN-γ及IL-4水平均显著高于正常对照。2.再障患者与正常对照骨髓造血细胞集落培养后CFU-GM计数再障患者BMMNCs在体外集落培养体系组（1）及组（2）中培养,第7天时CFU-GM计数分别为22.53±18.34和26.47±19.45（P＜0.05）,集簇计数分别为25.60±17.66和29.67±16.80（P＜0.05）。集落培养体系中加入α-Galcer后,有12例患者CFU-GM数目增加,有效率为80%（12/15）。正常对照BMMNCs在体外集落培养体系组（1）及组（2）中培养,第10天时CFU-GM计数分别为77.67±17.09和82.08±12.71（P＜0.05）。集落培养体系中加入α-Galcer后,也出现CFU-GM数目增加,有效率为75%（9/12）。结论:1.再障患者骨髓造血细胞液态扩增培养,其上清中IFN-γ及IL-4水平均显著高于正常对照。2.在骨髓造血细胞液态培养体系rhG-CSF+rhSCF中加入α-Galcer后,再障患者及正常对照培养上清中均出现IFN-γ水平显著下降,而IL-4水平显著升高。3.在骨髓造血细胞集落培养体系rhG-CSF+rhSCF中加入α-Galcer后,再障患者及正常对照均出现集落CFU-GM增加。更多还原

【Abstract】 Aplastic anemia(AA)is a syndrome of bone-marrow failure characterized by peripheral pancytopenia and marrow hypoplasia.Ontologically,threr are two kinds of aplastic anemia,namely,acquired AA and hereditary AA.Of these,acquired AA is the more common type.Acquired AA can be triggered by multiple factors including chemical agents such as exposure to toxic chemicals or chemotherapy drugs,physical agents such as ionizing radiation and biological agents mainly virus infections. However,in most of the cases,the cause of acquired AA is not known.Acquired AA is,therefore,referred to as idiopathic AA and abbrivated to AA throughourt the paper. Immunosuppressive therapy leads to remission in the majority of AA patients, suggesting that failure in hematopoiesis,at lease in some cases,is likely to be mediated by the aberrance in the immune system.Reduced CD4~+/CD8~+ ratio and elevated Th1/Th2 ratio are detected in both the blood and bone marrow of AA. Increased inhibitory cytokines,such as IFN-γ,TNF and IL-2 have also been detected. As a result,hematopietic stem/progenitor cells(HSPCs)in BM of AA patients are both quantitatively and qualitatively damaged by the abnormal cellular immunity and finally leads to BM failure.Natural killer T(NKT)cells are a unique T lymphocyte sub-population that has been implicated in the regulation of immune responses associated with a broad range of diseases,including autoimmunity,infectious diseases,and cancer.In human,NKT cells express a Vα24-Jα18-rearranged TCRαchain and a Vβ11-containingβchain,as well as NK cell-specific CD161.When activated,NKT cells result in a vigorous response evidenced by cytokine production within 1-2 hours.These cells release Th1-type cytokines including IFN-γ,and TNF,as well as Th2-type cytokines including IL-4 and IL-13.The mechanisms that determine the cytokine polarity of NKT cells,and the contribution of NKT cells to the systemic immune response,are not well understood,and indeed,this question represents one of the important challenges in the understanding of NKT cell roles in the regulation of cellular immunity.As an important immunoregulator in Th1/Th2 balance,NKT cells is, therefore,an attractable and interesting research area in the pathogenesis of AA.In the present study,we investigated the quantitative and qualitative changes of NKT cells in BMMNCs of AA patients in response to in vitro stimulation ofα-galactosylceramide(α-Galcer),and the effect of activated NKT cells on the expansion potential of HSPCs of AA patients.The objective of the study is to primarily look into the roles of NKT cells in the pathogenesis of AA,and to explore the possible application ofα-Galcer,as a new immuno-modulation agent,in the treatment of AA.PartⅠStudy of the Function of Expansion and Cytokine Releasing of TCRVα24~+Vβ11~+NKT Cells in Bone Marrow of Aplastic AnemiaObjective:To investigate the quantitative and qualitative changes of TCRVα24~+Vβ11~+NKT cells from BM of AA to the in vitro stimulation ofα-Galcer.Methods:TCRVα24~+Vβ11~+ NKT cells in the bone marrow mononuclear cells (BMMNCs)from either AA patients or healthy controls were enumerated with flow cytometry.BMMNCs were cultured in RPMI1640 medium supplemented with eitherα-Galcer and rhIL-2 orα-Galcer,rhIL-2 and rhG-CSE The proliferative capacity of NKT cells was expressed by fold expansion,as determined by NKT cell numbers after in vitro culture relative to those before the culture.Expression of intracellular IFN-γand IL-4 in activated NKT cells was analyzed with flow cytometry.Result:The percentage of NKT cells in BMMNCs from AA was significantly lower than that from healthy controls.Upon stimulation in vitro with eitherα-Galcer and rhIL-2 orα-Galcer,rhIL-2 and rhG-CSF,compared to those from healthy controls, NKT cells from AA showed reduced potential to expand,with a higher fraction of NKT cells expressing IFN-γ,(NKT1).When added to the culture medium,rhG-CSE in combination withα-Galcer and rhIL-2,decreased the expansion of NKT cells in both AA and control groups.It also induced polarization of NKT cells towards the NKT2 subpopulation,resulting in an elevated percentage of IL-4~+ NKT2 cells and a decreased percentage of IFN-γ~+ NKT1 cells in both AA and healthy control groups.Conclusion:Compared to those from healthy controls,BMMNCs from AA have a reduced fraction of NKT cells,which possesses a decreased potential to expand in vitro in response toα-Galcer stimulation,producing more IFN-γ~+ NKT1 cells. rhG-CSF,in combination withα-Galcer,confers polarization of NKT cells towards IL-4~+ NKT2 subpopulation.PartⅡStudy of the Effects of Activated NKT Cells on in vitro Hematopoietic Recovery in Aplastic AnemiaObjective:To investigate the effects of activated NKT cells on the expansion potential of HSPCs from AA.Methods:BMMNCs from AA patients and healthy controls were plated in IMDM medium supplemented with either rhSCF and rhG-CSF or rhSCF,rhG-CSF andα-Galcer.The level of cytokine IFN-γ/IL-4 in supernatant were determined by ELISA 3 days after culture.Colony-forming assay was conducted by culturing BMMNCs in methylcellulose medium supplemented with either rhSCF and rhG-CSF or rhSCF, rhG-CSF andα-Galcer.Yields of granulocyte/monocyte colony-forming unit (CFU-GM)were analyzed 7 days after plating.Result:Whenα-Galcer was added to the IMDM medium in combination with rhSCF and rhG-CSF,an elevated level of IL-4 and decreased level of IFN-γwere observed in supernatant in both AA and control groups.Meanwhile,with the addition ofα-Galcer to the methylcellulose medium,a significant increase in the number of CFU-GM was observed in both AA and control groups.Conclusion:An increased number of CFU-GM was generated in the BMMNCs from both AA and control groups whenα-Galcer was added in combination with rhG-CSF. These results indicate that NKT cells activated byα-Galcer and rhG-CSF are capable of modulating the abnormal T-cell mediated immunity and improving the in vitro expansion potential of HSPCs in AA.更多还原