NK细胞发育及蜕膜NK细胞在胚胎耐受中的功能

发布时间：2017-12-31 13:31

本文关键词：NK细胞发育及蜕膜NK细胞在胚胎耐受中的功能　出处：《中国科学技术大学》2011年博士论文　论文类型：学位论文

【摘要】：自然杀伤细胞(Natural killer cells,NK cells)是天然免疫系统的重要成员,分布于外周各淋巴器官及血液循环系统。不同与T、B细胞,NK细胞无需抗原的预先刺激与活化即可发挥细胞毒效应,并分泌多种细胞因子及趋化因子,具有杀伤微生物及招募免疫系统中其他细胞等多种生物学功能,从而在抗击感染,自身免疫病及肿瘤等多种疾病中发挥重要功能。近期越来越多的研究发现,NK细胞除杀伤肿瘤的能力外,更具有调节免疫系统和维持免疫平衡的功能。尤其是在妊娠早期,超过80%的母胎界面淋巴细胞均为NK细胞,而其免疫学功能至今不为人知。与此同时胎儿作为一种异基因移植物,却能够建立和维持妊娠的进行,这些都无法用经典的移植免疫耐受或肿瘤免疫逃逸理论来解释。因此,对NK细胞的调控功能,尤其对胚胎耐受的机理的研究,不仅对妊娠免疫极其重要,对研究移植免疫及临床NK细胞治疗也都有重要意义。与T细胞和B细胞相比,对NK细胞发育及其功能的认识至今仍然比较有限。为了更好的研究NK细胞在胚胎耐受中的作用,首先要了解蜕膜NK细胞的特征。蜕膜NK细胞在基因谱表达,发育阶段和功能上都与外周NK细胞有很大不同。为了进行NK细胞发育方面的研究,我们在研究中选取了三种不同组织来源的人的NK细胞:分别是外周血NK细胞,脐血NK细胞和蜕膜NK细胞。研究结果表明采用CD11b和CD27表面分子可以将人NK细胞划分为四个不同阶段。而蜕膜NK细胞与另外两种外周NK细胞相比,处于发育的早期。超过90%的外周血NK细胞都是CD11b+CD27-细胞亚群,而脐血NK细胞则80%是CD11b+CD27-NK亚群,20%是CD11b+CD27+NK亚群。有趣的是,研究中发现超过60%的人蜕膜NK细胞为CD11b-CD27-细胞亚群,这与外周NK细胞截然不同。这群CD11b-CD27-NK细胞表现出不成熟的特征,高表达NKG2A,并且具有向其他亚群细胞分化的潜能。从而更进一步验证了蜕膜NK细胞是处于发育早期的细胞群体。同时,本研究还证明了这四个不同亚群的NK细胞的功能也有所区别。CD11b-CD27+和CD11b+CD27+ NK细胞亚群在四个群体中分泌细胞因子的能力最强,CD11b+CD27-NK细胞主要表现为杀伤功能。因此,本研究表明不同组织来源的人NK细胞处于发育的不同阶段,并且具有不同的功能特征,从而建立了一种区分人NK细胞分化的新模型。同时,我们也验证了蜕膜NK细胞具有与外周NK不同的特性,包括有大量的具有发育分化潜能的CD11b-CD27-NK细胞、能分泌细胞因子的CD11b-CD27+和CD11b+CD27+NK细胞亚群。蜕膜NK细胞在发育上显示了与外周NK细胞所不同的独特特性,且在妊娠过程中又在母胎界面大量聚集,那么它在妊娠中行使的功能和相关的机制到底是怎样的呢?本研究发现在妊娠前三个月的蜕膜组织中有70%以上的淋巴细胞为NK细胞,而T细胞极少,低于10%。并且这些蜕膜NK细胞有20%以上的比例为CD56brightCD27+NK细胞,大大高于外周NK低于5%的该亚群比例。并且,随着妊娠的终结,晚期蜕膜局部的CD56brightCD27+NK细胞逐渐减少,伴随着T细胞增多,逐渐接近外周中的细胞比例。此外CD56brightCD27+NK细胞还是NK细胞中分泌IFN-γ等细胞因子的主要群体。那么只在妊娠早期,也就是胚胎植入最关键时间出现的CD56brightCD27+NK细胞究竟扮演了什么样的角色呢? 胎儿作为异基因移植物,在妊娠早期为了成功植入并生长,胎儿的滋养层细胞不停的入侵母体。在入侵的过程中必然引发一定的炎症反应。如何控制炎症反应,使之维持在温和平衡的范畴内,是胚胎耐受的关键问题。而TH17细胞,作为炎症过程中关键的致炎细胞,在很多疾病模型中发挥作用。我们检测了正常妊娠早期蜕膜组织中TH17细胞,发现正常妊娠中有2%以内的TH17细胞,显示了轻微的炎症。考虑到IFN-γ可以抑制TH17的极化,我们分别用外周血NK细胞和正常人蜕膜NK细胞证明,在正常妊娠中NK细胞可以通过分泌IFN-γ抑制TH17极化,从而维持胚胎耐受和免疫平衡。在因免疫原因反复流产病人中,我们发现其母胎界面局部有严重的炎症迹象,病理组织破损情况和浸润的TH17数量都有显著地上升。病人蜕膜NK细胞分泌抑制炎症的细胞因子IL-1RA等减少,NK比例下降,使得NK不能有效抑制TH17细胞极化。而与此同时,蜕膜中其他细胞却分泌大量IL-6和IL-1β等促进炎症因子,使炎症加剧。为了验证妊娠失败是否与TH17细胞的增加相关,我们还研究了自发流产经典模型CBA/J×DBA/2杂交小鼠的妊娠状态。结果显示,CBA/J×DBA/2孕鼠在妊娠第10天母胎界面与对照组相比有更多的TH17细胞浸润。进一步通过转输体外培养的TH17细胞,导致CBA/J×Balb/c小鼠也出现了胚胎吸收的流产现象。由此说明,大量的TH17的确会直接导致妊娠失败。综上所述,本研究证明CD11b和CD27标志可以将人的NK细胞划分为CD27-CD11b- NK细胞(DN NK细胞)、CD27+CD11b-NK细胞、CD27+CD11b+NK细胞(DP NK细胞)和CD27-D11b+NK细胞等四个具有不同功能的细胞亚群,蜕膜NK细胞处于发育的早期,并包含大量不成熟的DN NK(CD27-CD11b-)细胞。此外,本研究发现蜕膜NK细胞可以通过抑制炎性TH17细胞,维持母胎耐受和免疫平衡,并且这一过程是通过CD56brightCD27+NK细胞所分泌的IFN-γ和IL-1RA等细胞因子实现的。反复流产病人蜕膜组织中分泌的大量IL-6和IL-1β等炎症因子,促进了TH17细胞极化,导致炎症加剧;同时CD56brightCD27+NK细胞的比例下降,分泌IFN-γ减少,不能有效抑制TH17细胞的形成,母胎耐受平衡被打破,造成妊娠失败。本研究发现NK细胞能够作为调节性细胞抑制炎性反应,维持耐受平衡,保障妊娠正常进行的免疫学功能,对更好的理解NK细胞的调节功能,深入研究胚胎耐受的机理有重要意义。
[Abstract]:Natural killer cells (Natural killer cells, NK cells) is an important member of the innate immune system, distribution in the peripheral lymphoid organs and blood circulation system. Unlike T, B cells, NK cells to antigen without pre stimulus and activation can exert a cytotoxic effect, and the secretion of various cytokines and chemokines and with a variety of microorganisms and killing other cells in the immune system of recruitment and other biological functions, which play an important role in the fight against infection, autoimmune disease and tumors and other diseases. Recently more and more studies have found that NK cells in tumor killing ability, more can regulate the immune system and to maintain the immune balance function especially. In early pregnancy, more than 80% of the maternal fetal interface lymphocytes were NK cell, and its immune function is still unknown. At the same time as a fetal allograft was able to establish and Maintenance of pregnancy, these are not transplantation and tumor immune escape of the classical theory. Therefore, the regulation function of NK cells, especially the mechanism of embryo tolerance research, not only extremely important in pregnancy immunity, has important significance for the study of transplantation immunology and clinical NK cell therapy.
Compared with T cells and B cells, understanding of NK cell development and function is still relatively limited. In order to further study on the role of NK cells in the embryo tolerance, we must first understand the characteristics of NK cells in decidua. Decidual NK cells in the gene expression, development stages and functions and peripheral NK cells have great in order to carry out the research. The development of NK cells, we selected three kinds of human NK cells in the study are: NK cells in peripheral blood, umbilical cord blood NK cells and decidual NK cells. The results show that both CD11b and CD27 molecules on the surface of NK cells can be divided into four different stages and decidual NK cells compared with the other two peripheral NK cells in early development. More than 90% of the peripheral blood NK cells are CD11b+CD27- cell subsets and NK cells in cord blood of 80% CD11b+CD27-NK subgroups, 20% is CD11b+CD27+NK Group. Interestingly, the study found that more than 60% of human decidual NK cells of CD11b-CD27- cell subsets, the cells and peripheral NK are quite different. This group of CD11b-CD27-NK cells showed the characteristics of immature, high expression of NKG2A, and to other subsets of cell differentiation potential. In order to further verify decidual NK cells in the early development of the cell population. At the same time, this study also proves that these four different subsets of NK cell function also has the difference between.CD11b-CD27+ and CD11b+CD27+ NK cell subsets cytokine secretion in four groups in the strongest, mainly CD11b +CD27-NK cell killing function. Therefore, this study shows that NK cells derived from different tissues at different stages of development, and has the function of different characteristics, so as to establish a new model of human NK cell differentiation. At the same time, we also verified the decidual NK fine The cell has different characteristics from peripheral NK, including a large number of CD11b-CD27-NK cells with developmental differentiation potential, CD11b-CD27+ and CD11b+CD27+NK cell subsets that secrete cytokines.
NK cells in decidual development shows the unique characteristics of the peripheral NK cells are different, and during pregnancy in maternal fetal interface accumulation, so it exercise during pregnancy function and the relevant mechanism is what kind of? The study found in decidual tissues three months before pregnancy in more than 70% of the lymphocytes are NK cells and T cells are less than 10%. and the decidual NK cells have more than 20% of the proportion of CD56brightCD27+NK cells is much higher than that of peripheral NK is less than 5% of the subsets. And, with the end of late pregnancy, bureau of the Ministry of decidual CD56brightCD27+NK cells decreased gradually, with the number of T cells gradually, close to the peripheral cells in proportion. In addition the main groups of CD56brightCD27+NK cells or NK cells to secrete IFN- and other cytokines. So only in early pregnancy is the most critical during embryo implantation between What role does the CD56brightCD27+NK cell play?
As the fetal allograft in early pregnancy to successful implantation and growth of fetal trophoblast cells kept the invasion matrix. Inevitably bring certain inflammatory reaction in the invasion process. How to control the inflammatory reaction, to maintain a balance in the mild category, is a key problem of embryo tolerance. TH17 cells as the key, the inflammatory cells in the inflammatory process, play a role in many disease models. We detected TH17 cells of normal early pregnancy decidua, found that less than 2% of TH17 cells in normal pregnancy, shows a slight inflammation. Considering the IFN- gamma polarization can inhibit TH17, we used NK cells the peripheral blood and normal human decidual NK cells demonstrated that in normal pregnancy NK cells can secrete IFN- inhibited the TH17 polarization, thereby maintaining embryonic tolerance and immune balance.
For reasons of immune recurrent abortion patients, we found that the maternal fetal interface local severe signs of inflammation, tissue damage, and the number of pathological infiltration significantly increased TH17 patients. Decidual NK cells inhibit the inflammatory cytokine secretion of IL-1RA decreased, NK ratio decreased, so that NK can effectively inhibit TH17 cell polarization. At the same time, other cells in the decidua but the secretion of IL-6 and IL-1 beta promote inflammation, the inflammation aggravate.
In order to verify whether the pregnancy failure with the increase of TH17 cells related to pregnancy, we also study the classical model of spontaneous abortion CBA/J * DBA/2 hybrid mice. The results showed that CBA/J * DBA/2 pregnant rats on day tenth of pregnancy in maternal fetal interface compared with the control group had more TH17 cells. Further through the transfer of TH17 in vitro cells, leading to CBA/J * Balb/c mice also have abortion phenomenon. The embryo resorption, a large number of TH17 indeed will directly lead to pregnancy failure.
In summary, this study demonstrated that the CD11b and the CD27 logo can be divided into human NK cells NK CD27-CD11b- cells (DN NK cells), CD27+CD11b-NK cells, CD27+CD11b+NK cells (DP NK cells) and CD27-D11b+NK cells with four different functional cell subsets, NK cells in the decidua of early development, and contain a large number of immature DN NK (CD27-CD11b-) cells. In addition, these findings can be decidual NK cells through inhibition of inflammatory TH17 cells, maintenance of maternal fetal tolerance and immune homeostasis, and this process is realized by CD56brightCD27+NK cells by IFN- and IL-1RA and other cytokines. The secretion of large amounts of IL-6 and IL-1 beta and other inflammatory factors of recurrent spontaneous abortion the patient in the decidual tissue, promote TH17 cell polarization, lead to increased inflammation; at the same time, the proportion of CD56brightCD27+NK cells decreased, IFN- secretion decreased, the formation can not be effectively inhibited TH17 cells, Maternal fetal tolerance balance is broken, resulting in pregnancy failure. This study found that NK cells can act as regulatory cells inhibit the inflammatory response and maintain tolerance balance, guarantee the immunological function of normal pregnancy, to better understand the regulatory function of NK cells, have important significance to further study on the mechanism of embryo tolerance.

【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392.1

【共引文献】