【作者】 李莉；

【导师】 黄丽丽；

【作者基本信息】 浙江大学 ， 妇产科学， 2010， 博士

【摘要】 生殖免疫学的核心问题是母体对半同种异体移植物(胚胎)的耐受机制,这个谜团目前尚未被解开。既往的研究认为,母胎界面的局部免疫应答是母胎免疫耐受建立和维持的关键。母胎界面免疫微环境由子宫粘膜的免疫细胞及其分泌的细胞因子组成。目前对于子宫自然杀伤细胞(uterine natural killer cell, uNK)、T细胞在母胎免疫耐受中的作用研究比较深入,但对于树突状细胞(dendritic cells,DCs)在母胎免疫耐受中的作用研究甚少。DCs是机体内功能最强大的专职性抗原提呈细胞(antigen-presenting cell,APC),是特异性免疫应答的始动者,其激活T细胞的能力为巨噬细胞、B细胞的1000倍。人类DCs主要来源于骨髓CD34+造血干细胞(hematopoietic stem cells,HSC),广泛分布于机体粘膜表面,不仅能直接或间接激活T细胞、B细胞,引发机体免疫排斥,还能诱导免疫耐受,在机体对某些抗原免疫耐受的建立过程中发挥了重要作用。自从20世纪末DCs被正式确认以来,对DCs在免疫反应中的作用及机制的了解是近10年来免疫学领域最重大的进展之一。目前对DCs研究的热点是DCs在免疫激活及免疫耐受中的作用及机制。一般认为,成熟DCs诱导免疫激活,倾向于引发免疫排斥反应,不成熟DCs倾向于诱导机体免疫耐受。最新的研究证实人子宫内膜及蜕膜均含有DCs,在抵御外来病原体入侵及母胎免疫调控中起了重要作用。米非司酮是一种高效孕激素受体与糖皮质激素受体拮抗剂,近年来成功用于紧急避孕,其用药剂量在逐步减少。单次米非司酮剂量减到10mg,其避孕有效性仍很高,且不良反应少,对月经周期影响小,为目前通用的紧急避孕剂量。最新的研究发现低剂量米非司酮有望发展成一种新型的不抑制排卵的常规避孕药。每日给予低剂量米非司酮(1mg／d,稳态血药浓度65 nmol/L),不抑制排卵,但可使子宫内膜发育延迟,不利于胚胎着床而起到避孕效果,被称为“内膜避孕”,但其相关机制,尤其是免疫学机制研究甚少。本研究首先利用免疫组化技术观察DCs在人正常子宫内膜的分布及周期性变化,探索DCs在母胎免疫耐受中的作用。在成功制备人外周血来源DCs的基础上,在体外以不同浓度米非司酮作用于DCs,观察米非司酮对DCs成熟及生物学功能的影响,探索米非司酮作为内膜避孕药及紧急避孕药的免疫学机制。第一部分人正常子宫内膜DCs周期性变化研究目的：研究DCs在人正常子宫内膜的分布及周期性变化,探讨DCs在母胎免疫耐受中的作用。方法：40例正常子宫内膜样本取自具有正常月经周期的育龄女性,20例处于增生期(月经周期第6至10天),20例处于植入窗期(月经周期第20至24天)。常规HE染色判定组织学分期。免疫组化染色观察CD1a、CD83阳性细胞在子宫内膜的表达情况。结合血清雌二醇(E2)、孕酮(P)水平,分析DCs表达水平与E2、P的相关性。结果：1.人子宫内膜存在CD1a+DCs。植入窗期子宫内膜CD1a+DCs阳性率为100%,增生期子宫内膜CD1a+DCs阳性率为90%(18／20)2.人子宫内膜DCs数量随月经周期变化而变化,植入窗期CD1a+DCs细胞密度为18.2±5.76 cells/mm2,显著高于增生期子宫内膜CD1a+DCs细胞密度(6.5±4.05 cells/mm2, P＜0.05)。3.子宫内膜DCs数量与血清孕激素水具有相关性(相关系数为0.630,P<0.01)。4.未发现CD83+成熟DCs存在于人正常子宫内膜。结论：1.人子宫内膜存在DCs,为未成熟DCs。2.人子宫内膜DCs数量随月经周期变化而变化,受卵巢性激素调节。3.植入窗期子宫内膜未成熟DCs数量显著上升,这可能有利于胚胎顺利着床,诱导母胎耐受。第二部分人外周血来源DCs的体外扩增及鉴定目的：从健康人外周血中分离和纯化单核细胞,体外培养诱导分化为DCs,为后续实验奠定基础。方法：淋巴细胞分离液密度梯度离心法从健康成年女性外周血浓缩白细胞分离获得单个核细胞。免疫磁珠分选CD14+单核细胞。纯化后的CD14+单核细胞在含有100 ng/ml rhGM-CSF、20 ng/ml rhIL-4的RPMI1640培养基中培养6天分化成为未成熟DCs。加入100 ng/ml LPS继续培养48小时诱导DCs成熟。相差显微镜及扫描电镜观察细胞形态。流式细胞仪分析细胞免疫表型。混合淋巴细胞反应(mixed leukocyte reactions, MLR)检测DCs刺激同种异体T淋巴细胞增殖的能力。结果：1.单核细胞体外培养6天后,相差显微镜、扫描电镜观察到细胞呈现典型DCs形态。2.新鲜分离的单核细胞表型为CD14+CD1a-。体外培养6天后成功分化为未成熟DCs,流式细胞术分析表型为CD14-CD1a+HLA-DRlowCD83-。在不同的刺激细胞／反应细胞比例下(DC/T 1:125,1:25, 1:5),MTT去测定OD值分别为0.1123,0.1975,0.3665,即刺激T细胞增殖的能力较弱。3.培养第8天的细胞(LPS促成熟48小时),显著上调表达HLA-DR及CD83分子(P<0.05)。在不同的刺激细胞／反应细胞比例下(DC／T 1：125,1：25,1：5),OD值分别为0.1563,0.2825,0.5180,与培养第6天的细胞相比,具有很强的刺激同种异体T淋巴细胞增殖的能力(P<0.05),其刺激能力随着刺激细胞／反应细胞比例(DC／T)升高而增强。结论：人外周血CD14+单核细胞经GM-CSF和IL-4体外培养获得大量DCs。细胞形态、免疫表型及混合淋巴细胞反应证实其为典型DCs。第三部分米非司酮对人外周血来源DCs成熟和功能的影响目的：在成功制备人外周血来源DCs的基础上,在体外以不同浓度米非司酮作用于DCs,观察米非司酮对DCs成熟及生物学功能的影响,探索米非司酮作为抗着床避孕药及紧急避孕药的免疫学机制。方法：人外周血CD14+单核细胞在体外经GM-CSF、IL-4培养6天诱导分化为未成熟DCs。加入不同剂量的米非司酮(20 nmol/L,65 nmol/L,200 nmol/L和1800 nmol/L)继续培养48小时,同时设置阳性对照组(加入100ng/ml LPS),阴性对照组及溶剂对照组(加入米非司酮溶剂dimethyl sulphoxide, DMSO)。流式细胞仪检测和分析细胞表面CDla.CD83和HLA-DR分子的表达情况。酶联免疫吸附测定(enzyme linked immunosorbnent assay, ELSIA)检测DCs培养上清液中IL-12p70水平。MLR检测各组DCs刺激同种异体T细胞增殖的能力。结果：1.米非司酮上调DCs表面HLA-DR及CD83分子表达。2.与阴性对照组相比,米非司酮处理组DCs分泌的IL-12p70水平显著升高(P<0.05)。在一定范围内(20 nmol/L～200 nmol/L),米非司酮促进DCs分泌IL-12p70的能力随着米非司酮浓度增大而增强。3.与阴性对照组相比,米非司酮处理组DCs刺激同种异体T细胞增殖的能力明显增强(P<0.05)。在一定范围内(20 nmol/L～200 nmol/L),米非司酮促进DCs刺激T细胞增殖的能力随着米非司酮浓度增大而增强。结论：1.米非司酮诱导DCs表型成熟,促进DCs分泌IL-12并增强其刺激T淋巴细胞增殖的能力。2.在一定浓度范围内,米非司酮对DCs的促成熟作用存在量效关系。3.米非司酮可能通过促进DCs成熟,引发母体对半同种异体胎儿抗原免疫排斥,达到抗着床避孕。4.米非司酮紧急避孕的免疫学机制与内膜避孕可能不完全相同。更多还原

【Abstract】 The survival of the semi-allogeneic fetus has been an immunological paradox. The mechanism by which the fetus is not rejected by maternal immune system is unclear. It is generally accepted that fetal survival depends on the local immunity at the maternal-fetal interface. Human endometrium is richly populated by maternal leukocytes, including uterine naturally killer (uNK) cell, T cell, macrophage and DCs. Considerable studies have been focused on the role of uNK and T cell in maternal-fetal tolerance. Recent evidence suggests that DCs are critical mediators of immunological interactions at the maternal-fetal interaction.DCs are the most potent antigen-presenting cells with a unique ability to induce primary immune responses. The capacity of DCs to activate T cells is 1,000 times more than macrophages and B cells. Human mucosal surfaces contain specialized DCs capable of sensing these external stimuli and mounting appropriate local responses depending on the nature of the elements they encounter. Emerging evidence indicates that DCs are responsible for the establishment of tolerance as well as immunity. Generally, immature DCs induce tolerance and mature DCs induce immunity. Although small numbers in endometrium and decidua, recent evidence suggests that DCs are involved in this balance between immune defence and maternal-fetal tolerance.Mifepristone (Ru486), a potent progesterone and glucocorticoid receptor antagonist, is widely used as an emergency contraceptive and its dosage is gradually reduced. A single dose of 10 mg mifepristone is one of the most effective hormonal methods for emergency contraception, with little side effects. Recently, mifepristone has shown the potential to be used as a novel estrogen-free contraceptive which disturbs endometrial development enough to prevent implantation without affecting ovulation. Daily dose of 1 mg mifepristone, reaching a steady plasma level of 65 nmol/L with no impairment on ovulation, was effective to prevent pregnancy and is called "endometrium contraception". But the exact mechanism of endometrial contraception, especially immunological mechanism, remains unknown.In current study, we first investigated endometrial dendritic cell populations during the normal menstrual cycle by immunohistochemistry and explored the role of DCs in the establishment of maternal-fetal tolerance. Then we investigated the effect of mifepristone on the phenotypic and functional maturation of human moncyte-derived DCs, and consequently, to elucidate the mechanism of mifepristone as an anti-implantation contraceptive or emergency contraceptive.PartⅠEndometrial dendritic cells populations during the normal menstrual cycleObjective:To investigate the number and distribution of dendritic cells in normal endometrium of reproductive age during the normal menstrual cycle. And to explore the role of DCs in the establishment of maternal-fetal tolerance. Methods:Normal endometrial sample weres collected from forty women of reproductive age,20 in the proliferative phase (d6-10) and 20 in the "window of implantation" (d20-24). Endometrial specimens were stained with hematoxylin and eosin for histological examination. And the expression of CD1a and CD83 was evaluated by immunohistochemistry.Results:1. CD1a positive dendritic cells were found in all cases of the "window of implantation" and most cases of the proliferative phase (18/20,90%).2. The density of CD1a+DCs in the "window of implantation" was 18.2±5.76 cells/mm2, significantly higher than that in the proliferative phase (6.5±4.05 cells/mm2, P＜0.05).3. Serum progesterone level was correlated with the density of CD1a+ DCs.4. No CD83+ DCs were found in human emdometrium.Conclusion:1. Human endometrium contains CD1a+ DCs, but no CD83+ mature DCs.2. The number and maturation of DCs may be regulated by ovarian hormones.3. The highly coordinated cyclical changes in DCs populations may be important for the establishment of maternal-fetal tolerance. PartⅡExpansion of DCs from human peripheral blood in vitroObjective:To generate large numbers of DCs from CD14+ monocytes in vitro.Methods:Peripheral blood mononuclear cells (PBMC) from healthy female donors (buffy coats obtained from the local central blood bank) were isolated by Ficoll-Hypaque density gradient separation. CD14+ monocytes were enriched with bead-labeled anti-CD 14 mAb on a high-gradient magnetic cell sorting system (MACS). Purified CD14+ monocytes were cultured in PRMI1640 medium containing 100 ng/ml rhGM-CSF and 20 ng/ml rhIL-4 for 6days. LPS (100 ng/ml) was added for another 48 hours to induce maturation. The morphology was monitored by light microscopy and scanning electron microscopy, and the immunophenotypes were determined by flow cytometry. Allogeneic mixed leukocyte reactions were used for immune functional assays.Results:1. Light microscopy and scanning electron microscopy showed typical DCs morphology after 6 days.2. Freshly isolated monocytes were CD14+CD1a-.After 6 days, cells showed an immature DCs phenotype (CD14-CD1a+ HLA-DRlowCD83-) and induced little or no allogeneic T cells proliferation in MLR.3. Addition of LPS to immature DCs resulted in a marked increase in the expression of HLA-DR, as well as CD83, indicative of DCs maturation. And mature DCs stimulated allogeneic T cell proliferation markedly in a stimulator/responder ratio-dependent manner. Conclusion: This study developed a simple way to generate large numbers DCs from PBMC with rhGM-CSF and rhIL-4. With analyzing morphology, phenotypes and function, we confirmed these cells were typical DCs.PartⅢMifepristone induces maturation of human monocyte-derived dendritic cellsObjective:To investigate the effect of mifepristone on the phenotypic and functional maturation of human moncyte-derived DCs, and consequently, to elucidate the mechanism of mifepristone as an anti-implantation contraceptive or emergency contraceptive.Methods:DCs were generated from human peripheral blood CD14+ cells cultured with GM-CSF and IL-4. On day 6, immature DCs were left untreated (negative control), stimulated with various concentrations of mifepristone (20 nmol/L,65 nmol/L,200 nmol/L and 1800 nmol/L), DMSO (solvent control), or LPS (posotive control,100 ng/mL). After 48 hours, DCs were harvested and stained with mAbs against CD1a, CD83 and HLA-DR to determine their phenotype by flow cytometric analysis. The cytokine levels secreted by DCs were determined by ELISA and the allostimulatory capacity of DCs was measured by MLR.Results:1. Flow cytometric analysis revealed that mifepristone upregulated the surface expression of MHC classⅡmolecules and specific molecule CD83.2. Compared with negative control, mifepristone enhanced the secretion of IL-12p70 by DCs significantly (P＜0.05). Up to 200 nmol/L, the IL-12p70 production by DCs was upregulated by mifepristone in a dose-dependent manner.3. Compared with negative control, mifepristone-treated DCs strongly stimulates the proliferation of allogeneic T cell in a mixed lymphocyte reaction (P＜0.05). Up to 200 nmol/L, the capacity of stimulating the proliferation of T cell was upregulated by mifepristone in a dose-dependent manner.Conclusion:Mifepristone induced a distinct phenotypic and functional maturation of DCs in vitro. Similar mechanisms may be effective in vivo, providing an explanation for the possible mechanism of mifepristone as a contraceptive agent, especially as an anti-implantation contraceptive.更多还原