【作者】 肖敏；

【导师】 凌斌；

【作者基本信息】 安徽医科大学 ， 妇产科学， 2008， 硕士

【摘要】 研究背景:妊娠期肝内胆汁淤积症(ICP)是妊娠晚期特有的并发症,以全身皮肤瘙痒、肝功能异常、伴有不同程度的黄疸为特征,其主要危及胎儿,易引起早产、胎儿窘迫、死胎、死产等。目前对于ICP确切的发病原因,以及宫内缺氧的机制尚未完全明确,迫切需要明确阐述ICP的发病原因,才有可能建立有效的预防、治疗措施。已有研究表明该病的发生与妊娠期母体免疫功能紊乱有关。近年来国内外提出了母胎界面这一概念,其核心内容是母胎界面由母体的子宫蜕膜和胎儿滋养层共同构成,母胎界面内的多种细胞成份及其分泌的细胞因子构成了特殊的母胎界面免疫微环境,在保护胎儿生长发育、免受母体的免疫排斥中起重要作用。在人类妊娠过程(尤其早期妊娠)中,NK细胞是蜕膜中最丰富的淋巴细胞,它与胚胎滋养层细胞直接接触,是唯一粘附于滋养层细胞表面的子宫淋巴细胞。dNK细胞主要分布于母体与胚胎接触的蜕膜,是母体直接识别胎儿抗原的免疫细胞,它的独特的表型及功能不但能维持母胎界面的免疫耐受,还能分泌一系列细胞因子促进蜕膜血管重建及Th1/ Th2平衡网络的维持。若dNK细胞的数量及功能失调则可能诱发病理妊娠。本课题研究ICP患者蜕膜及外周血中NK细胞的含量、表型、功能状态的变化,研究ICP患者蜕膜及外周血中NK细胞的Th1、Th2型细胞因子的分泌情况是否同时出现在局部免疫与系统免疫,为进一步研究母胎免疫耐受的形成机制、ICP患者母胎界面免疫微环境变化的细胞学及分子生物学机制提供良好的实验基础。探讨ICP患者全身系统与母胎界面局部免疫微环境变化的关系,无疑为ICP的发病机制提供新的解释。目的:检测ICP患者蜕膜及外周血中NK细胞的含量、表型、Th1/Th2型细胞因子表达、杀伤功能方面的变化,探讨ICP患者蜕膜及外周血中NK细胞独特的表型、功能及其与滋养细胞的相互识别关系在系统免疫与母胎界面免疫耐受之间的重要联系。方法:1)收取重度ICP患者(实验组,15例)和正常晚孕(对照组,15例)壁蜕膜组织及同一人外周血,机械研磨法联合密度梯度离心法分离出蜕膜单个核细胞,单纯密度梯度离心法分离出外周血单个核细胞,流式细胞术检测蜕膜及外周血中NK细胞的含量及表型。2)收集重度ICP患者和正常晚孕胎盘组织各6例,RT-PCR技术检测两组胎盘滋养层中NKG2A的配体HLA-G, HLA-E和NKG2D的配体MICA mRNA的表达情况。3) ELISA法检测ICP组和正常晚孕组壁蜕膜及外周血单个核细胞上清中IFN-γ和IL-4的表达4)流式细胞术检测ICP组和正常晚孕组壁蜕膜及外周血中NK细胞内Th1/Th2型细胞因子IFN-γ和IL-4的表达。5) LDH法分别测定蜕膜及外周血中单个核细胞对NK细胞敏感性靶细胞K562的杀伤活性。结果:1)外周血中的NK细胞是以CD56+CD16+为主,蜕膜中的NK细胞是以CD56+CD16-为主。ICP组外周血中的NK细胞含量明显高于对照组外周血中的NK细胞含量(P<0.05),ICP组蜕膜中NK细胞含量明显高于对照组蜕膜中的NK细胞含量(P<0.05)。ICP组外周血中CD3-CD56+细胞、CD56+CD16-细胞、CD56+CD16+细胞、CD56+NKG2D+细胞含量均显著升高(P<0.05),而CD56+NKG2A+细胞细胞含量无显著变化(P>0.05)。ICP组蜕膜中CD3-CD56+细胞、CD56+CD16-细胞、CD56+CD16+细胞、CD56+NKG2D+细胞含量均显著升高(P<0.05),而CD56+NKG2A+细胞含量无显著变化(P>0.05)。2)与对照组相比,ICP组蜕膜NK细胞高表达NKG2受体家族NKG2A和NKG2D,但在两组滋养层中检测到NKG2A的配体HLA-G和HLA-E,而未检测到NKG2D的配体MICA,而且没有差别。3)与对照组相比,ICP患者外周血及蜕膜单个核细胞培养上清中INF-γ含量均显著增高(P<0.05),而IL-4含量无显著变化(P>0.05)。4)与对照组相比,ICP患者dNK及pNK细胞内Th1/ Th2型细胞因子IFN-γ和IL-4的表达均明显增高(P<0.05)。5)在一定效靶比情况下,ICP组蜕膜及外周血单个核细胞对NK细胞敏感性靶细胞K562的杀伤活性明显高于对照组蜕膜及外周血单个核细胞。结论:ICP组外周血及蜕膜组织中NK细胞在含量、表型、Th1/Th2细胞因子表达及杀伤功能方面均与正常晚孕对照组存在差异,并与滋养层细胞之间有密切联系。异常的免疫应答不仅出现在ICP患者的全身系统,而且突出表现在母胎界面局部。蜕膜NK细胞独特的表型及功能在介导母胎界面免疫耐受中发挥重要作用。更多还原

【Abstract】 Backgroud:Intrahepatic cholestasis of pregnancy (ICP) is thought to a complication of late pregnancy, with character of all over the body Itch of skin, liver disfunction, distinct degree morbus arcuatus. It is associated with an increased risk of fetal distress, causing premature deliveries, fetal distress, fetal deaths and stillbirths. Presently, the cause of ICP and the mechanisms of oxygen deficiency of infants are not fully elucidated, no drug treatment has achieved the ideal goals. Only after etiological factor and mechanisms of placental hypofunction are identified completely, effective measure of precaution and therapy may be designed.Though studies suggest that the disturbance of maternal immune system function may relate to the pathogenesis of the disease, its etiology and pathogenesis are unknown. Recent research on maternal-fetal interface has been proposed, the main content is composed of maternal uterus deciduas and fetal trophoblast, all kinds of cells and secreted cytokine are composed of special immunological microenvironment on at the maternal-fetal interface, which is crucial for embryo implantation and gestational maintenance. In early pregnancy, NK cells are enriched at sites where fetal trophoblast infiltrates the decidua. The appearance of decidual NK cells suggest that one of the functions of these cells is control of placentation. Histologic studies indicate NK cells are in direct contact with the invading trophoblast by adhesion molecules CD56. Decidual NK cells mainly distribute in decidua which links fetal and maternal interface and which can directly recognize fetal antigen. The specific phenotype and function of dNK cells may contribute to keep immunotolerance in fetal-maternal interface, also can they secrete an array of cytokines facilitate decidual blood rebuilding and maintain the balance of Th1/ Th2 cytokines. To study the functions and status of dNK cells undoubtedly not only helps us to comprehend the physiological phenomena of pregnancy and but also to provide new ways to research the mechanism of pathological pregnancy, as well as the development in reproduction immunology.None of the aforementioned studies refered to the change of percentage, phenotype, function and Th1/Th2 cytokine balance in peripheral blood natural killer (pNK) cells and decidua natural killer (dNK) cells in the patients with ICP. To further shed light on the immunological mechanisms of ICP, we focus our attention on local immune reactions at the maternal-fetal interface and system immunity reactions between normal late pregnant women (NLP) and ICP. We study the percentage and the phenotype in the decidua natural killer (dNK) cells in the patients with ICP, investigate the change of Th1/Th2 cytokine levels of decidual mononuclear cells and peripheral blood mononuclear cells in pregnant women with ICP, to futher investigate the change of Th1/Th2 cytokine levels in peripheral blood natural killer (pNK) cells and decidua natural killer (dNK) cells, so that we can investigate whether disturbed local immune reactions and system immunity reactions are also present in pregnant women with ICP. Undoubtedly, Elucidation of immunologic microenvironment at maternal-fetal interface with ICP must be an original pathway to investigate the mechanism of ICP.Objective:To detect the change of dNK cells and pNK cells, to compare the percentage, phenotype, Th1/ Th2 cytokine secretion and cytotocxic action between dNK cells and pNK cells between normal late pregnant women (NLP) and ICP, which can help us to further discover the important interaction between dNK cells and trophoblast cells immunotolerance local immune reactions at the maternal-fetal interface and system immunity. Methods:1) The same pregnant women third-trimester parietalis decidua and peripheral blood were obtained during elective caesarean sections (CS) in ICP patients (n= 15) and NLP (n=15). Peripheral blood mononuclear cells (PBMC) were centrifugated using a standard gradient sedimentation technique cell suspensions were prepared by an electromechanical dispersal method and centrifugated using a standard gradient sedimentation technique. And percentage and phenotype of dNK cells were detected by flow cytometric analysis.2) To detect the mRNA expression of HLA-G, HLA-E and MICA at trophoblasts from elective caesarean sections (CS) in ICP patients (n=6) and NLP (n=6) by the method of RT-PCR. They are ligands of NKG2A and NKG2D respectively.3) The concentration of Th1-type cytokine (INF-γ) was increased in patients suffering from ICP by enzyme linked immunosorbent assay, Whereas no differences were found in the level of IL-4.4) To detect the expression of intracellular Th1/Th2 cytokines IFN-γand IL-4 in dNK cells and pNK cells by the flow cytometry.5) The cytotoxic activity of dNK cells and pNK cells against targets was measured in by the LDH assay.Results:1) Compared to controls, the parietalis deciduas from ICP patients were characterized with the increased the percentage of CD3-D56+NKcells, CD56+CD16+ NK cells, CD56+CD16- NK cells, CD56+NKG2D+ NK cells. Whereas no differences were found in the percentage of CD56+NKG2A+cells.2) Decidual natural killer cells had high expression of NKG2A, which ligand HLA-G and HLA-E mRNA were also expressed in trophoblasts tissue. Although dNK cells highly expressed NKG2D, there was no MICA mRNA expression.3) The concentration of Th1-type cytokine (INF-γ) was significantly increased in patients suffering from ICP. Whereas no differences were found in the levels of IL-4.4) The expression of intracellular Th1/Th2 cytokines IFN-γand IL-4 were significantly high in dNK cells in ICP pregnant women. IFN-γand IL-4 are the most important cytokines in regulating Th1/Th2 immunological balance, suitable quantity of which play an important role in regulating immunological balance at fetal-maternal interface5) Their cytotoxic activity of dNK cells and pNK cells in ICP team against targets was significantly increased than those of dNK and pNK cells cells in NLP team.Conclusions:Decidual NK cells and peripheral NK cells in ICP team are the important natural lymphocytes, whose percentage, phenotype, Th1/Th2 cytokine secretion and cytotocxic activity are obviously different from decidual NK cells and peripheral NK cells NLP team, and also they tightly touch with the invading trophoblasts. dNK cells and their secreting cytokines compose the center of decidual immunological regulation through development, regulation, receptor expression, biological effect. Abnormal immune responses not only appear insystem immunity but also mainly manifest fetal-maternal interface immunity. From the above results, we conclude that dNK cells play an important role in regulating immunological tolerance at fetal-maternal interface and system immunity更多还原