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  本文關(guān)鍵詞：NK細胞發(fā)育及蛻膜NK細胞在胚胎耐受中的功能　出處：《中國科學技術(shù)大學》2011年博士論文　論文類型：學位論文

  更多相關(guān)文章： 人NK細胞 CD11b/CD27 細胞發(fā)育 胚胎耐受 母胎界面 蛻膜組織 免疫平衡

【摘要】：自然殺傷細胞(Natural killer cells,NK cells)是天然免疫系統(tǒng)的重要成員,分布于外周各淋巴器官及血液循環(huán)系統(tǒng)。不同與T、B細胞,NK細胞無需抗原的預先刺激與活化即可發(fā)揮細胞毒效應,并分泌多種細胞因子及趨化因子,具有殺傷微生物及招募免疫系統(tǒng)中其他細胞等多種生物學功能,從而在抗擊感染,自身免疫病及腫瘤等多種疾病中發(fā)揮重要功能。近期越來越多的研究發(fā)現(xiàn),NK細胞除殺傷腫瘤的能力外,更具有調(diào)節(jié)免疫系統(tǒng)和維持免疫平衡的功能。尤其是在妊娠早期,超過80%的母胎界面淋巴細胞均為NK細胞,而其免疫學功能至今不為人知。與此同時胎兒作為一種異基因移植物,卻能夠建立和維持妊娠的進行,這些都無法用經(jīng)典的移植免疫耐受或腫瘤免疫逃逸理論來解釋。因此,對NK細胞的調(diào)控功能,尤其對胚胎耐受的機理的研究,不僅對妊娠免疫極其重要,對研究移植免疫及臨床NK細胞治療也都有重要意義。 與T細胞和B細胞相比,對NK細胞發(fā)育及其功能的認識至今仍然比較有限。為了更好的研究NK細胞在胚胎耐受中的作用,首先要了解蛻膜NK細胞的特征。蛻膜NK細胞在基因譜表達,發(fā)育階段和功能上都與外周NK細胞有很大不同。為了進行NK細胞發(fā)育方面的研究,我們在研究中選取了三種不同組織來源的人的NK細胞:分別是外周血NK細胞,臍血NK細胞和蛻膜NK細胞。研究結(jié)果表明采用CD11b和CD27表面分子可以將人NK細胞劃分為四個不同階段。而蛻膜NK細胞與另外兩種外周NK細胞相比,處于發(fā)育的早期。超過90%的外周血NK細胞都是CD11b+CD27-細胞亞群,而臍血NK細胞則80%是CD11b+CD27-NK亞群,20%是CD11b+CD27+NK亞群。有趣的是,研究中發(fā)現(xiàn)超過60%的人蛻膜NK細胞為CD11b-CD27-細胞亞群,這與外周NK細胞截然不同。這群CD11b-CD27-NK細胞表現(xiàn)出不成熟的特征,高表達NKG2A,并且具有向其他亞群細胞分化的潛能。從而更進一步驗證了蛻膜NK細胞是處于發(fā)育早期的細胞群體。同時,本研究還證明了這四個不同亞群的NK細胞的功能也有所區(qū)別。CD11b-CD27+和CD11b+CD27+ NK細胞亞群在四個群體中分泌細胞因子的能力最強,CD11b+CD27-NK細胞主要表現(xiàn)為殺傷功能。因此,本研究表明不同組織來源的人NK細胞處于發(fā)育的不同階段,并且具有不同的功能特征,從而建立了一種區(qū)分人NK細胞分化的新模型。同時,我們也驗證了蛻膜NK細胞具有與外周NK不同的特性,包括有大量的具有發(fā)育分化潛能的CD11b-CD27-NK細胞、能分泌細胞因子的CD11b-CD27+和CD11b+CD27+NK細胞亞群。 蛻膜NK細胞在發(fā)育上顯示了與外周NK細胞所不同的獨特特性,且在妊娠過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關(guān)的機制到底是怎樣的呢?本研究發(fā)現(xiàn)在妊娠前三個月的蛻膜組織中有70%以上的淋巴細胞為NK細胞,而T細胞極少,低于10%。并且這些蛻膜NK細胞有20%以上的比例為CD56brightCD27+NK細胞,大大高于外周NK低于5%的該亞群比例。并且,隨著妊娠的終結(jié),晚期蛻膜局部的CD56brightCD27+NK細胞逐漸減少,伴隨著T細胞增多,逐漸接近外周中的細胞比例。此外CD56brightCD27+NK細胞還是NK細胞中分泌IFN-γ等細胞因子的主要群體。那么只在妊娠早期,也就是胚胎植入最關(guān)鍵時間出現(xiàn)的CD56brightCD27+NK細胞究竟扮演了什么樣的角色呢? 胎兒作為異基因移植物,在妊娠早期為了成功植入并生長,胎兒的滋養(yǎng)層細胞不停的入侵母體。在入侵的過程中必然引發(fā)一定的炎癥反應。如何控制炎癥反應,使之維持在溫和平衡的范疇內(nèi),是胚胎耐受的關(guān)鍵問題。而TH17細胞,作為炎癥過程中關(guān)鍵的致炎細胞,在很多疾病模型中發(fā)揮作用。我們檢測了正常妊娠早期蛻膜組織中TH17細胞,發(fā)現(xiàn)正常妊娠中有2%以內(nèi)的TH17細胞,顯示了輕微的炎癥�？紤]到IFN-γ可以抑制TH17的極化,我們分別用外周血NK細胞和正常人蛻膜NK細胞證明,在正常妊娠中NK細胞可以通過分泌IFN-γ抑制TH17極化,從而維持胚胎耐受和免疫平衡。 在因免疫原因反復流產(chǎn)病人中,我們發(fā)現(xiàn)其母胎界面局部有嚴重的炎癥跡象,病理組織破損情況和浸潤的TH17數(shù)量都有顯著地上升。病人蛻膜NK細胞分泌抑制炎癥的細胞因子IL-1RA等減少,NK比例下降,使得NK不能有效抑制TH17細胞極化。而與此同時,蛻膜中其他細胞卻分泌大量IL-6和IL-1β等促進炎癥因子,使炎癥加劇。 為了驗證妊娠失敗是否與TH17細胞的增加相關(guān),我們還研究了自發(fā)流產(chǎn)經(jīng)典模型CBA/J×DBA/2雜交小鼠的妊娠狀態(tài)。結(jié)果顯示,CBA/J×DBA/2孕鼠在妊娠第10天母胎界面與對照組相比有更多的TH17細胞浸潤。進一步通過轉(zhuǎn)輸體外培養(yǎng)的TH17細胞,導致CBA/J×Balb/c小鼠也出現(xiàn)了胚胎吸收的流產(chǎn)現(xiàn)象。由此說明,大量的TH17的確會直接導致妊娠失敗。 綜上所述,本研究證明CD11b和CD27標志可以將人的NK細胞劃分為CD27-CD11b- NK細胞(DN NK細胞)、CD27+CD11b-NK細胞、CD27+CD11b+NK細胞(DP NK細胞)和CD27-D11b+NK細胞等四個具有不同功能的細胞亞群,蛻膜NK細胞處于發(fā)育的早期,并包含大量不成熟的DN NK(CD27-CD11b-)細胞。此外,本研究發(fā)現(xiàn)蛻膜NK細胞可以通過抑制炎性TH17細胞,維持母胎耐受和免疫平衡,并且這一過程是通過CD56brightCD27+NK細胞所分泌的IFN-γ和IL-1RA等細胞因子實現(xiàn)的。反復流產(chǎn)病人蛻膜組織中分泌的大量IL-6和IL-1β等炎癥因子,促進了TH17細胞極化,導致炎癥加劇;同時CD56brightCD27+NK細胞的比例下降,分泌IFN-γ減少,不能有效抑制TH17細胞的形成,母胎耐受平衡被打破,造成妊娠失敗。本研究發(fā)現(xiàn)NK細胞能夠作為調(diào)節(jié)性細胞抑制炎性反應,維持耐受平衡,保障妊娠正常進行的免疫學功能,對更好的理解NK細胞的調(diào)節(jié)功能,深入研究胚胎耐受的機理有重要意義。
[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.

【學位授予單位】：中國科學技術(shù)大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R392.1

【共引文獻】

相關(guān)期刊論文 前2條

1 許園園;劉雨生;;樹突狀細胞在妊娠免疫中的研究進展[J];國際生殖健康/計劃生育雜志;2009年01期

2 吳志勇;程明軍;吳玉;徐從劍;;DKK1在早期自然流產(chǎn)患者蛻膜中的表達[J];現(xiàn)代婦產(chǎn)科進展;2010年01期

相關(guān)碩士學位論文 前2條

1 許園園;外周血調(diào)節(jié)性T淋巴細胞及Foxp3的表達與反復體外受精—胚胎移植失敗的相關(guān)性研究[D];安徽醫(yī)科大學;2009年

2 錢志大;復發(fā)性流產(chǎn)婦女蛻膜CD83~+、CD1a~+樹突狀細胞的研究[D];浙江大學;2010年

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本文編號：1360009