【作者】 班艳丽；

【导师】 孔北华；

【作者基本信息】 山东大学 ， 妇产科学， 2008， 博士

【摘要】 妊娠的成功恰恰依赖于母胎免疫耐受状态,但其中的机制至今尚不十分清楚。妊娠早期母胎界面成分及其构成的特殊的免疫微环境对母胎免疫耐受的维持至关重要。位于母胎界面的蜕膜被认为是一个免疫特赦组织,妊娠早期大量白细胞在此选择性聚集,其中最主要的是蜕膜NK（decidual natural killer,dNK）细胞,约占淋巴细胞总量的70-80%,另外还有树突状细胞（dendritic cells,DCs）、单核巨噬细胞和T细胞。这些免疫活性细胞在局部组织微环境的调控下能够获得特异性的表型和功能,从而决定局部免疫反应的取向-免疫应答或免疫耐受形成。研究报道吲哚胺2,3-双加氧酶（indoleamine 2,3-dioxygenase,IDO）广泛表达于妊娠早期的母胎界面,它是肝脏以外唯一可催化色氨酸（L-tryptophan）分子中吲哚环氧化裂解,从而经犬尿氨酸（L-kynurenine）途径代谢的限速酶,调控色氨酸代谢。IDO的活性表达诱导形成“色氨酸耗竭微环境”,使细胞处于一种色氨酸饥饿状态,通过抑制T细胞及NK细胞增殖和诱导致耐受性DCs形成等多种机制在免疫反应调控中发挥着关键作用。研究表明妊娠时胎盘滋养细胞产生的IDO能够抑制母体T淋巴细胞反应,从而保护胎儿对抗母体的排斥反应,而体内试验加入IDO抑制剂1-甲基色氨酸（1-methyl-tryptophan,1-MT）可导致同种异基因胎儿排斥反应。可见正常的IDO表达和活性是维持妊娠所必需的,然而母胎界面IDO表达对蜕膜NK细胞及DCs的影响还不清楚。蜕膜NK细胞具有独特的表型和功能特点,是妊娠早期一道独特的风景。对其表型分析结果显示:90%的uNK细胞为CD56^brightCD16^-,而90%的外周血NK（peripheral-blood NK,pNK）细胞为CD56^dimCD16⁺。dNK细胞是妊娠早期母胎界面数量最多的淋巴细胞,主要分布于母体与胚胎接触的底蜕膜并与侵入的滋养层细胞密切接触,它可能是母体直接识别胎儿抗原的免疫细胞,在维持母胎免疫平衡和促进早期胎盘生长发育中发挥着重要作用。dNK细胞功能状态是影响母胎界面免疫调控的关键因素,而NK细胞表面活化性受体或抑制性受体激活后发出的活化性信号或抑制性信号的平衡调节决定着dNK细胞的功能状态。研究发现dNK细胞高表达能够介导dNK细胞杀伤功能的自然杀伤活性受体（naturalcytotoxicity receptors,NCRs）,如NKp46、NKp44、NKp30和NKG2D,另外还高表达穿孔素和颗粒酶。这些研究提示dNK细胞具有潜在的杀伤活性。值得注意的是正常妊娠时dNK杀伤活性较低。但在某些病理妊娠如复发性自然流产（recurrent spontaneous abortion,RSA）时,这些活化性受体的表达和功能发生了变化,从而使dNK细胞杀伤功能增强进而导致妊娠排斥。可见,正常妊娠时dNK细胞可能受到母胎界面免疫微环境的调控而维持低杀伤活性,但其机制还不清楚。最近有报道IDO的代谢产物犬尿氨酸能够特异性抑制人外周血来源的NK细胞表面自然杀伤活性受体NKG2D和NKp46的表达,从而抑制NK细胞的杀伤活性,并且还能够减少NK细胞IFN-γ和TNF-α等细胞因子的分泌。另有研究报道犬尿氨酸还能够抑制NK细胞增殖。然而母胎界面活性表达的IDO对dNK细胞和pNK细胞表型和功能的影响还不清楚。推测IDO可以调控dNK细胞表面活化性受体的表达并影响其杀伤活性功能,从而诱导免疫耐受。新近,蜕膜DCs亚群被认为在维持母胎免疫调控中发挥着关键作用。DCs是功能最强的抗原提呈细胞,不同的DCs亚群和表型特点可能控制免疫反应的强度和性质,从而能够决定局部免疫反应的取向-免疫应答或免疫耐受形成。DCs功能受局部组织微环境的调控并与其表面抗原表达谱有关。大量研究证实DCs可以通过上调IDO表达而诱导免疫耐受。有研究报道蜕膜Lin^-HLA-DR⁺DCs能够表达IDO。这些研究提示母胎界面IDO表达参与蜕膜DCs功能的调控。但是由于缺乏特异性标志物以及数量较少,以往对对蜕膜DCs的研究受到极大的限制,目前蜕膜DC亚群的分布及其表型特点还不十分清楚。而研究妊娠早期蜕膜组织中DCs亚群及其表型特点,将为进一步研究IDO对蜕膜DCs功能的影响提供理论基础。本研究分为三部分,第一部分主要探讨妊娠早期母胎界面IDO表达情况以及与复发性自然流产的关系;第二部分探讨IDO是否调控dNK细胞表面杀伤活性受体表达和杀伤活性等功能;第三部分是利用新近发现的BDCA标志来检测早孕期蜕膜组织中DCs亚群及其表型特点,并进一步分析DCs亚群的数量随孕周变化的特点。本研究有助于深入理解母胎免疫耐受调节机制,以期为母胎免疫失衡相关妊娠并发症的诊治提供新的策略。第一部分妊娠早期母胎界面及滋养细胞株HTR-8/SVneo中IDO表达与复发性自然流产的关系目的:探讨人妊娠早期母胎界面及人绒毛外细胞滋养细胞株HTR-8/SVneo中IDO的表达与RSA的关系。方法:（1）免疫组化法检测正常妊娠早期和RSA患者绒毛和蜕膜组织中IDO蛋白质表达;定量RT-PCR法检测两组绒毛和蜕膜组织中IDO mRNA的表达。（2）绒毛组织块培养法培养人妊娠早期绒毛组织,高效液相色谱（HPLC）法检测培养上清中有无犬尿氨酸,并分析IFN-γ对IDO功能活性的影响。（3）免疫细胞化学法检测HTR-8/SVneo细胞中IDO蛋白质表达;定量RT-PCR法检测IDOmRNA表达水平;HPLC法检测培养上清中有无犬尿氨酸,并分析IFN-γ对IDO表达水平及功能活性的影响。结果:（1）免疫组化法发现IDO主要表达于绒毛合体滋养细胞、绒毛外细胞滋养细胞以及蜕膜腺上皮细胞,少数病例可见IDO主要表达于细胞滋养细胞和绒毛外细胞滋养细胞,而绒毛合体滋养细胞未见有表达;IDO mRNA在绒毛组织和蜕膜组织中均有表达,（2）绒毛组织块培养法培养人妊娠早期绒毛,方法简单,成功率高,培养48小时后发现绒毛组织表达的IDO具有功能活性,并且IFN-γ刺激组IDO活性显著高于单纯加DMEM/F12培养组（P＜0.05）。（3）HTR-8/SVneo细胞有蛋白质及IDO mRNA表达;细胞培养上清中可检测到犬尿氨酸;IFN-γ可以上调IDO蛋白质及mRNA表达,呈药物浓度的依赖性（P＜0.05）。（4）RSA组绒毛和蜕膜组织中IDO蛋白质及mRNA表达水平均显著低于正常妊娠组（P＜0.05）。结论:绒毛及蜕膜组织IDO正常表达是维持妊娠所必需;HTR-8/SVneo细胞表达有活性的IDO。第二部分HTR-8/SVneo细胞IDO表达对dNK细胞和pNK细胞表面NKG2D和NKp46表达及杀伤活性功能的影响目的:探讨HTR-8/SVneo细胞IDO表达对dNK细胞和pNK细胞表面活化性受体NKp46和NKG2D表达及杀伤活性等功能的影响。方法:（1）体外分离正常孕5-9周蜕膜组织单个核细胞和外周血单个核细胞,免疫磁珠分选法纯化CD56⁺dNK细胞和pNK细胞。（2）FCM法检测分离纯化的CD56⁺dNK细胞和pNK细胞的纯度和表型。（3）将分离纯化的CD56⁺dNK细胞和pNK细胞体外培养48小时,根据条件培养基（CM）类型将试验分为3组:正常培养基组（阴性培养基对照组）;HTR-8/SVneo细胞48h获得的条件培养基组（IDO CM组）;HTR-8/SVneo细胞+色氨酸阻断剂1-MT 48 h获得的条件培养基组（IDO+1-MT CM组）。培养48小时后收集细胞,采用定量RT-PCR法分别检测CD56⁺dNK细胞和pNK细胞表面受体NKp46 mRNA及NKG2D mRNA表达水平,FCM法分析NKp46及NKG2D蛋白质表达水平。（4）进一步采用LDH法检测CD56⁺dNK细胞和pNK细胞对靶细胞的杀伤活性。（5）ELISA法检测培养上清中的TNF-α水平。结果:（1）机械法体外分离正常5-9孕周蜕膜组织单个核细胞,Ficoll-Hypaque法分离蜕膜单个核细胞和外周血单个核细胞,免疫磁珠分选法进一步纯化CD56⁺dNK细胞和pNK细胞。（2）FCM法分析免疫磁珠分选法纯化的CD56⁺CD3^-NK细胞纯度,结果显示:占dNK细胞的90%以上,其中CD56^brightCD16^dim占97%以上;占pNK细胞的55%以上,其中CD56^dimCD16^-NK细胞占40%左右,CD56^dimCD16⁺NK细胞占60%左右。（3）dNK细胞分组试验结果显示,阴性培养基对照组、IDO CM组和IDO+1-MT CM组组间比较,NKG2D和NKp46 mRNA和蛋白质表达水平没有显著差异;pNK细胞分组试验结果显示,NKG2D和NKp46 mRNA和蛋白质表达水平在IDO CM组显著高于阴性培养基对照组和IDO+1-MT CM组（P＜0.05）。（4）dNK细胞杀伤活性在阴性培养基对照组、IDO CM组和IDO+1-MT CM组各组间比较无显著差异（P＞0.05）;pNK细胞杀伤活性在IDO CM组显著低于阴性对培养基照组和IDO+1-MTCM组（P＜0.05）。（5）dNK细胞培养上清中的TNF-α水平各组间比较无显著差异（P＞0.05）;pNK细胞培养上清中TNF-α水平在IDO CM组显著低于阴性培养基对照组和IDO+1-MT CM组（P＜0.05）。结论:HTR-8/SVneo表达的IDO能够下调pNK细胞表面NKp46及NKG2D受体表达,并降低pNK杀伤活性和分泌TNF-α的能力,而不降低dNK细胞杀伤活性等功能。第三部分妊娠早期蜕膜组织中BDCA-1（+）,BDCA-2（+）和BDCA-3（+）树突状细胞的分布和表型特点目的:利用新近发现的BDCA标志来检测妊娠早期蜕膜组织中DC亚群及其表型特点,并进一步分析DC亚群数量随孕周变化的特点。方法:（1）机械法分离6-9孕周（n=44,每孕周11例）蜕膜组织中的单个核细胞,FCM法检测蜕膜单个核细胞中BDCA-1⁺ MDC1,BDCA-3⁺ MDC2和BDCA-2⁺ PDC亚群。（2）FCM法进一步检测BDCA-1⁺ MDC1和BDCA-3⁺ MDC2亚群的表型特点,分析HLA-DR、CD86、CD80和ILT3的表达水平。（3）免疫组织化学法检测妊娠早期蜕膜组织（n=12）中BDCA-1⁺和BDCA-3⁺细胞。结果:（1）发现正常妊娠早期蜕膜组织中存在3个DC亚群,分别是:BDCA-1⁺CD19^-CD14^-MDC1.BDCA-3⁺CD14^-MDC2和BDCA-2⁺CD123⁺PDC亚群。蜕膜组织中MDC1占分离的蜕膜单个核细胞的百分率与外周血相似,两组比较无统计学差异（P=0.055）。蜕膜组织中MDC2显著高于外周血,而PDC显著低于外周血（P＜0.001）。（2）MDC1与MDC2均表达低水平的HLA-DR,CD86和CD80,提示它们具有未成熟DCs的表型特点;MDC1与MDC2均表达ILT3。（3）随着孕周增加（6-9周）,蜕膜单个核细胞中MDC1比例显著降低,而MDC2的比例显著增加（P＜0.05）。（4）免疫组化法发现妊娠早期蜕膜组织中存在BDCA-1⁺和BDCA-3⁺细胞,BDCA-3⁺细胞分布于近血管和近腺体周围。结论:我们首次描述了妊娠早期蜕膜组织中存在BDCA-1⁺CD19^-CD14^-MDC1,BDCA-3⁺CD14^-MDC2和BDCA-2⁺CD123⁺PDC亚群。它们的分布和表型特点提示其可能参与母胎免疫耐受的诱导。本研究为进一步了解DCs在母胎界面免疫调节中的作用提供了理论依据。更多还原

【Abstract】 In human pregnancy,the implanted embryo constitutes a hemiallograft but remains spared from attack by the maternal immune system,suggesting a tolerance of its fetus at the site of contact between mother and child.Serving as an immunologically privileged tissue,the decidua and its components,especially decidual leucocytes,play essential functions in pregnancy maintenance.The decidual leucocyte population has been a centre of interest for the understandingof the mechanism that might control maternal immune responses in successful pregnancy. During the first trimester of pregnancy,the human decidua is rich in leucocytes which make up 10-15%of all decidual cells.This leucocyte population is composed of 70% decidual natural killer（dNK）cells,10%T cells and 20%major histocompatibility complex（MHC）classⅡ-positive antigen-presenting cells（APCs）which are thought to be mainly macrophages and dendritic cells（DCs）Indoleamine 2,3-dioxygenase（IDO）has been found in placenta,decidual dendritic cells,monocytes and glandular epithelium.IDO is a heme-containing rate-limiting enzyme,which catalyzes the conversion of tryptophan to kynurenine,the main tryptophan metabolite.Tryptophan is the least available essential amino acid and is required by all forms of life for protein synthesis and other important metabolic functions.IDO-generated low tryptophan environment may inhibit T cell and NK cells proliferation and induce tolerogenic DCs,and play a key role in pregnancy maintance.In 1998,Munn et al.showed that murine placenta is protected from immune rejection by maternal T cells by means of localized IDO dependent depletion of tryptophan.Following work from the same group suggested that rejection of the allogeneic fetus is accompanied by a unique form of inflammation that is characterised by T cell dependent and antibody independent activation of complement. IDO synthesis is necessary to maintain effective immunological protection during gestation.While the mechanisms of IDO-dependent inhibition of T-cell function have been elucidated,less is known on the possible role of IDO in regulating natural killer （NK）-cell activity and DCs functions.Decidual NK cells has unique phenotypical and functional characteristcs,and the phenocyte of 90%Decidual NK cells is CD56^birghtCD16^- and less than 10%of dNK is CD56^dimCD16⁺.Human CD56^birghtNK cells accumulate in the maternal decidua during first trimester of pregnancy and are found in direct contact with fetal trophoblasts.It is currently believed that in the early stages of gestation,dNK cells may play an important role in the control of immune tolerance at the fetal-maternal interface and trophoblastic growth,differentiation,and invasion.NK-cell function is regulated by a balance between activating and inhibitory signals.Decidual NK cells can express natural cytotoxicity receptors（NCRs）,such as NKp46、NKp44、NKp30 and NKG2D.Transcriptional geneexpression profiling has shown that dNK cells express both perforin and granzymes at a similar or even higher level than CD56^dim CD 16⁺ pNK cells.These observations suggested that dNK cells might have cytotoxic potential.During normal pregnancy,dNK cells remain cytotoxicity at a low level. However,abnormal expression and function of activating recepors may lead to pregnancy loss.It has recently been shown that the tryptophan catabolite L-kynurenine inhibits the surface expression of NKp46-and NKG2D-activating receptors and regulates NK-cell function.Also IDO-generated L-kynurenine,in addition to T-cell proliferation,is also able to inhibit the IL-2-induced proliferation of NK cells.The effect of IDO expressed at the fetal-maternal interface is still largely unknown.We formulated the hypothesis that IDO expression at the fetal-maternal interface may inhibit the surface expression of NKp46-and NKG2D-activating receptors and regulate NK-cell function.To study the mechanism that control the cytotoxicity of dNK cells may help to understand the mechanism of immune tolerance at the fetal-maternal ineraction.Interestingly,the human decidua was described recently to harbour DCs,which has pointed to a critical role of DCs at the fetal-maternal interface.DCs can acquire unique features or phenotypes in different tissue microenvironments,and different DC subsets may play a prominent role in dictating the quantity and quality of immune responses and decide whether immunity or tolerance develops.Accumulating evidence suggests that DCs in situ can induce antigenspecific unresponsiveness or tolerance in central lymphoid organs and in the periphery.DCs can induce maternal tolerance by up-regulating IDO expression.It was reported that IDO expression in decidual monocytes and DCs by CTLA-4/Fc or IFN-γtreatment were increased in therapeutic abortion decidua but were decreased in spontaneous abortion,suggesting the key role of IDO expression on DCs.In the past,the investigation of DC subsets in the human decidua has been hampered by the lack of specific markers identifying DCs directly and by the scarcity of DCs.Several groups have reported the presence of certain DC subsets at the fetal-maternal interface;however,the precise distributional and phenotypic characteristics of DC subsets in the human decidua are still poorly understood.The study to the effect of IDO expression on the function of dNK Cells and the distribution and phenotypes of decidual DCs will provide new clue for understanding of physical and pathological reproduction and new thread for diagnosis and therapy of reproductive immune disease.PARTⅠRELATIONSHIP OF RECURRENT SPONTANEOUS ABORTION AND THE INDOLEAMINE 2,3-DIOXYGENASE EXPRESSION AT THE FETAL -MATERNAL INTERFACE AND HTR-8/SVNEO CELLSObjective:To study the relationship of recurrent spontaneous abortion（RSA） and the expression of IDO in villi,decidua and HTR-8/SVneo cells in vitro.Methods:（1）Immunohistochemistry was applied to analyze the expression of IDO protein in villus and decidua from normal pregnancy and RSA.Quantitative RT-PCR was applied to analyze the mRNA transcription of IDO.（2）Human trophoblast villous explant was cultured,and highperformance liquid chromatography （HPLC）was applied to determinate whether there was kynurenine in culture medium of trophoblast villous explant and the effect of IFN-γon the mRNA expression and activity of IDO.（3）Immunohistochemistry was applied to analyze the expression of IDO protein in HTR-8/SVneo cells.Quantitative RT-PCR was applied to analyze the mRNA transcription of IDO.HPLC was applied to determinate whether there was kynurenine in culture medium of cells and the effect of IFN-γon the mRNA expression and activity of IDO.Results:（1）IDO protein was expressed in villous syncytiotrophoblast,EVT cell columns and decidual gland.In minor patients,IDO protein was expressed in cytotrophoblast and EVT cell column,but syncytiotrophoblast.IDO mRNA was detected in villus and decidua.（2）Human trophoblast villous explant ultured for 48 h was used to determine IDO activity by HPLC.The level of IDO activity treated with IFN-γwas significantly higher than that of control（P＜0.05）.（3）IDO protein and mRNA were found to be expressed in HTR-8/SVneo cells,and there was kynurenine in the cell culture medium.（4）The expression of IDO protein and mRNA in villus and decidua from RSA were lower than that from normal pregnancy.Conclusion:Appropriate expression of IDO at the fetal-maternal interface is necessary for maintenance of normal pregnancy,and an active IDO protein was expresseed in HTR-8/SVneo cells.PARTⅡTHE EFFECT OF INDOLEAMINE 2,3-DIOXYGENASE EXPRESSED IN HTR-8/SVNEO CELLS ON THE SURFACE EXPRESSION OF NATURAL CYTOTOXICITY RECEPTORS AND THE FUNCTION OF DECIDUAL NK CELLSObjective:To study the effect of IDO expressed in HTR-g/SVneo cells on the surface expression of NKG2D and NKp46 and the cytotoxicity of dNK cells and dNK cells.Methods:（1）Decidual mononuclear cells were isolated from 5-9 weeks’ decidua obtained from clinically normal pregnancies in vitro.CD56⁺ dNK cells were purified by use of human CD56 MicroBeads.（2）Purity and phenotype of CD56⁺ NK cells were then analyzed by flow cytometry（FCM）.（3）CD56⁺dNK cells were cultured for 48 hours with complete RPMI 1640 medium,IDO conditioned medium（CM）which were obtained from HTR-8/SVneo cells,and IDO + 1-MT CM which were obtained from HTR-8/SVneo cells.Then qRT-PCR and FCM were applied to analyze the mRNA transcription and protein expression of NKG2D and NKp46 on dNK cells and pNK cells.（4）The cytotoxicity of dNK cells and pNK cells obtained were assessed by LDH assays.（5）The culture supernatants were then collected and analyzed for the presence of TNF-αby using ELISA.Results:（1）CD56⁺ dNK cells were purified by use of human CD56 MicroBeads. （2）The purity of dNK cells was all over 95%.The phenotype of dNK cells was CD56^bright.（3）The expression of IDO mRNA and protein on CD56⁺ pNK cells cultured with IDO CM were significantly lower than the other two groups（P＜0.05）. （4）The pNK cells cultured with IDO showed a redudced ability to kill K562 cells than the other two groups（P＜0.05）.（5）The level of TNF-αin IDO group was significantly lower than that in the other two groups（P＜0.05）.Conclusion:IDO expressed in HTR-8/SVneo cells was found to prevent the cytokine-mediated up-regulation of the expression and function of NKG2D and NKp46 responsible for the induction of NK-cell-mediated killing.PARTⅢBDCA-1（+）,BDCA-2（+）AND BDCA-3（+） DENDRITIC CELLS IN EARLY HUMAN PREGNANCY DECIDUAObjective:The aim of this study was to identify and characterize DCs in the cell isolates obtained from normal human first-trimester decidua with the recently developed BDCA markers.Whether the number of these DC subsets changes with gestational age was also investigated. Methods:（1）A non-enzymatic method was used to isolate decidual mononuclear cells from 6-9 weeks’ decidua（n = 44）obtained from clinically normal pregnancies. Flow cytometry was used to analyse the cell preparations in terms of staining for BDCA-1,CD14,CD19,BDCA-3,CD14,BDCA-2 and CD123.（2）To characterize further the phenotypes of fresh decidual MDC1 and MDC2,flow cytometric analysis was performed with a panel of MoAbs including HLA-DR,CD80,CD86 and Immunoglobulin-like transcript 3（ILT3）.（3）Immunohistochemical staining was used to analyze the distribution of BDCA-1⁺ and BDCA-3⁺ cells in the decidua（n = 12）.Results:（1）Using flow cytometry,we identified three DC subsets in normal human first-trimester decidua:BDCA-1⁺ CD19^- CD14^- myeloid DC type 1（MDC1）, BDCA-3⁺ CD14^- myeloid DC type 2（MDC2）and BDCA-2⁺ CD123⁺ plasmacytoid DC（PDC）.The percentage of MDC1 to mononuclear cells in the decidua was similar to that in the peripheral blood controls.The percentage of MDC2 in the decidua was significantly higher than that in the peripheral blood controls,whereas the percentage of PDC was significantly lower.（2）Both MDC1 and MDC2 subsets expressed HLA-DR,CD86 and CD80 at low levels,suggesting a characteristic of immature myeloid DCs.ILT3,suggested to be involved in immune tolerance induction,was also expressed on decidual MDC1 and MDC2 subsets.（3）In addition,as gestational age increased from 6 to 9 weeks,the numbers of MDC1 decreased but MDC2 increased significantly.（3）BDCA-1⁺ cells are present in the decidual stroma,BDCA-3⁺ cells are present in the decidual stroma near maternal vesses and glands.Conclusions:we have demonstrated the presence of the three previously unidentified BDCA+ DC subsets in normal human first-trimester decidua: BDCA-I⁺CD19^-CD14^-MDC1,BDCA-3⁺ CD14^- MDC2 and BDCA-2⁺ CD123⁺ PDC. The distributional and phenotypic characteristics of these decidual DC subsets may be relevant to the immune tolerance necessary for the maintenance of pregnancy. Therefore,this study provides a basis for further research of the roles of DCs in immunoregulation of human pregnancy at the fetal-maternal interface.更多还原