人脾脏基质细胞诱导新型调节性树突状细胞亚群的产生及其相关机制研究

发布时间：2018-01-04 13:38

本文关键词：人脾脏基质细胞诱导新型调节性树突状细胞亚群的产生及其相关机制研究　出处：《第二军医大学》2010年硕士论文　论文类型：学位论文

【摘要】： 树突状细胞(Dendritic cell, DC)被认为是目前已知的功能最强大的专职抗原提呈细胞(antigen presenting cells, APC),能够感知、整合一系列的外来病原体信号并将其传递给淋巴细胞,启动适度的免疫应答反应以清除入侵机体的病原体。未成熟DC在微生物感染或移植后识别、吞噬外源性抗原,可快速成熟。DC成熟后吞噬能力减弱,但细胞表面共刺激分子和粘附分子的表达上调,并分泌促炎症细胞因子,启动初始的T细胞介导的免疫应答反应。近年来,越来越多的研究表明DC是一种异质性的细胞群体,分布于不同的解剖部位,含有不同的细胞亚群,处于不同的成熟阶段,表达不同的表型和细胞因子,其功能也是多样性的。其中,具有负向免疫调控作用的DC亚群的发现及其功能特点与作用机制的研究是该领域近年来的重要进展之一,此类具有负向免疫调控作用的DC亚群被称为调节性DC(Regulatroy DC).目前认为,调节性DC主要通过选择性的诱导Th2型的免疫应答或促使初始CD4+和CD8+T细胞分化为分泌IL-10的调节性T细胞来负向调控免疫应答。在前期的研究中,以小鼠为实验对象,我们发现以往被认为终末分化细胞的成熟DC,与小鼠脾脏基质细胞共培养后能够进一步增殖并分化为一类能够通过分泌NO而抑制T细胞增殖的新型调节性树突状细胞,我们将之命名为“分化样树突状细胞”(Differentiated dendritic cell, diffDC),从而提出了成熟DC并非均为终末分化细胞的观点,其在完成了抗原递呈任务之后,在次级淋巴器官微环境的作用下,仍能够进一步增殖分化,从而被赋予了新的生命与功能。在不同的生理或病理情况下,diffDC与周围很多免疫细胞存在相互作用,彼此精密调节,例如可以通过分泌IL-10活化NK细胞而促进其杀伤能力,并通过分泌IP-10选择性地趋化Thl细胞并抑制其增殖,从而维持机体的稳态。然而,上述观点都是来自于针对小鼠的实验结果,在人体内成熟树突状细胞与脾脏基质微环境又是如何作用的呢?是否会产生像小鼠diffDC那样的具有负向调节功能的人树突状细胞亚群呢?如果真有这样一种人DC亚群,其生物学意义及其相关的作用机制又是什么呢? 鉴于本课题本质为两种细胞的相互作用,我们在本研究中分为三部分内容,分别围绕脾脏基质细胞系的建立、新型人调节性树突状细胞亚群的特点与功能以及该细胞亚群的人体内验证三方面进行探讨。一、人脾脏基质细胞系HESSCs的建立与鉴定首先,我们分离4-6月引产胎儿新鲜脾脏,用组织培养法培养脾脏基质细胞。大约一个月左右,贴壁生长的基质细胞铺满培养板,消化传代后形成最初的脾脏基质细胞系。此时的脾脏基质细胞系在镜下观察可见由数种形态、大小不一的细胞组成,纯度不佳。随着进一步的培养,逐步纯化为主要由内皮状细胞组成的基质细胞系,细胞生长速度趋于稳定,如此经过大约2-3月的培养后,形成稳定的脾脏基质细胞系。接下来,我们对所建的人类脾脏基质细胞系进行了初步鉴定,通过流式检测分析发现该细胞系主要是由CD105+的内皮细胞组成,不表达CD11c、CDllb等髓系细胞标志。mRNA分析表明该基质细胞系以内皮型细胞为主,检测通过ELISA检测发现该细胞系分泌较高的TGF-p,提示该细胞系可能具有一定的诱导分化能力,因此我们构建的脾脏基质细胞系是一群主要由内皮细胞构成的具有潜在诱导分化能力的基质细胞系,我们称其为HESSCs (Human Endothelial-like Spleen Stromal Cells) 二、HESSCs诱导产生的新型人调节性树突状细胞亚群的特点与功能在本部分实验中,我们着重研究了由人脾脏基质细胞系HESSCs诱导的,从人外周血单核细胞来源的成熟树突状细胞分化而来的CD11bhiCTLA-4+新型人调节性树突状细胞亚群的特点与功能。首先,我们从正常人外周血样本分离得到人外周血单个核细胞(PBMC),进一步纯化为CD14+单核细胞,用GM-CSF及IL-4联合诱导其分化为典型的未成熟树突状细胞(imDC),再用TLR配体LPS刺激其分化为成熟树突状细胞(maDC)后,将该maDC与之前建立的脾脏基质细胞系HESSC混合培养,观察二者之间的相互作用。实验发现,去除诱导性细胞因子GM-CSF及IL-4后,对照培养基中的maDC大部分在1-2周内凋亡或死亡,而大部分与HESSCs混合培养的树突状细胞依旧存活,说明HESSCs支持单核细胞来源的maDC的存活。基于上述现象,我们选定与HESSC共培养1-2周后的maDC作为研究对象,进行表型与功能方面的检测。经流式分析发现这些细胞相对于maDC主要表现为CD11clow、CD11bhi、HLA-DRlow、CD80low、CD83low、CD86low及CTLA-4+,同时细胞因子检测证实该群细胞高分泌PGE2、TGF-p,然而目前尚未发现该群细胞能够分泌较高的IL-10、NO等负向调节因子。令人感兴趣的是,该群细胞表达抑制性受体CTLA-4,加之其又能分泌高水平的PGE2、TGF-p等负向调节细胞因子,提示该群新型树突状细胞亚群可能发挥着一定的免疫抑制功能。通过对maDC及该新型树突状细胞亚群的mRNA进行分析对比,验证了该群新型树突状细胞亚群TGF-β、CTLA-4的mRNA表达水平都明显高于maDC,同时尚未发现该树突状细胞亚群的IL-10、PD-1、PDL-1、PDL-2等重要分子的mRNA水平有明显增高。树突状细胞作为最主要的抗原提呈细胞,在炎症的发生发展过程中发挥着重要的调控作用,那么我们这群新型的树突状细胞亚群在炎症状态下又有怎样的反应呢?实验证明,该新型树突状细胞亚群与maDC相比在TLR4配体LPS刺激后,能够分泌高水平的炎性细胞因子,包括IL-10、TNF-α、PGE2、TGF-β等。这一结果提示我们该新型树突状细胞亚群可能在炎症状态下发挥着更大的免疫调节作用。由HESSCs诱导的这群新型调节性树突状细胞表达CTLA-4,而CTLA-4作为重要的负向调节分子,在抑制T细胞免疫应答过程中发挥重要作用,因此我们推测该新型树突状细胞亚群可能具有免疫抑制的功能。通过体外混合淋巴反应实验,我们发现该新型DC亚群确实能够显著抑制maDC刺激后异体CD4+T细胞的增殖,具体的机制还有待于进一步研究。众所周知,DC在未成熟状态下具有极强的吞噬能力,当其遇到刺激或吞噬抗原分化成熟后吞噬能力就基本丧失。而由成熟DC分化而来的该新型DC亚群的吞噬能力却又大大增强,这一现象为我们对其功能的深入研究探讨提供了极大的想象空间。总之通过上述实验,发现脾脏基质细胞系HESSCs可以诱导外周血单核细胞来源的maDC分化为一种高分泌PGE2、TGF-β等调节性因子的具有独特表型CD1 1bhiCTLA-4+的新型人调节性树突状细胞亚群。我们初步将其命名为CD11bhiCTLA-4+DCreg。三、人体内新型调节性树突状细胞亚群的鉴定在本部分研究工作中,我们着重在人体脾脏内寻找并初步验证了这类新型调节性树突状细胞的存在。为了验证该新型DC亚群是否真的存在于体内,我们在人脾脏中寻找该DC亚群。我们首先用该新型DC亚群的特异性表面标志(CD11c+CD11bhiCTLA-4+)将表型相同的细胞分选出来,再通过检测它的细胞因子分泌情况及其抑制功能来判断人体内是否存在这样一群还未被人们发现的DC亚群。我们用流式分选仪将人体脾脏内CD11c+细胞分选为CD11chiHLA-DRhiCD11blow、CD11chiHLA-DRhiCD11bhi和CD11cintHLA-DRlowCD11bhi三个细胞亚群来研究,经过对细胞表型、混合淋巴反应、细胞因子分泌及吞噬能力多个方面进行检测对比,证实人体脾脏内存在一群CD11chiHLA-DRhiCD11bhi的细胞表达CTLA-4,且分泌大量的PGE2、TGF-β、IL-10等细胞因子,具有较强的吞噬能力,在所占比例较大的情况下可以有效抑制体外maDC诱导的异体CD4+T淋巴细胞的增殖。因此我们初步判断人脾脏内CD11chiHLA-DRhiCD11bhi细胞与体外脾脏基质细胞系HESSCs诱导分化的CD11bhiCTLA-4+DCreg具有相似的特点与功能。综上所述,我们通过实验发现,人脾脏基质细胞系HESSCs可以诱导人外周血单核细胞来源的成熟DC分化为一群具有负向免疫调节作用的新型人树突状细胞亚群。该细胞亚群具有独特的细胞表型CD11bhiCTLA-4+,高分泌PGE2、TGF-β等负向调节性因子,能够有效抑制CD4+T淋巴细胞的增殖,并初步验证了其在人体(脾脏)内的存在。这些实验结果丰富了对免疫微环境负向调控免疫应答多细胞网络的认识,为今后进一步研究树突状细胞参与免疫负向调控的机制提供了新的方向和一定的实验基础,为探索肿瘤、自身耐受和自身免疫性疾病等的发生机制及免疫治疗的应用提供了新的研究途径。
[Abstract]:Dendritic cells (Dendritic cell DC) is considered to be a professional antigen known as the most powerful antigen-presenting cells (antigen, presenting, cells, APC) is able to perceive the integration of a wide array of incoming signals and delivers them to lymphocytes, start an immune response reaction free moderate to eliminate invading pathogens of immature DC. In the recognition of microbial infection or after transplantation, phagocytosis of exogenous antigen,.DC can quickly mature mature phagocytic ability is abate, but cell surface costimulatory molecules and up-regulated the expression of adhesion molecules, and proinflammatory cytokines, start the immune response of primary T cell-mediated. In recent years, more and more studies show that DC is a heterogeneous cell population distribution in different anatomic sites, containing different subpopulations of cells in different stages of maturity, and the phenotypic expression of different cytokines, its function It is also diversity. Among them, the research has a negative effect to the immune regulation of DC subgroup discovery and function characteristics and mechanism of action is one of the important progress in recent years in the field, this has a negative effect to the immune regulation of DC subsets called regulatory DC (Regulatroy DC). At present, regulation DC mainly through the immune response induced by Th2 selective or to the initial CD4+ and CD8+T cells to secrete IL-10 regulatory T cells to negatively regulate immune response.
In a previous study, mice as experimental subjects, we found previously considered mature DC cell terminal differentiation, coculture with mouse splenic stromal cells can further proliferate and differentiate into a class could inhibit the proliferation of T cells by secreting NO and new regulatory dendritic cells, which we named "the differentiation of dendritic cells" (Differentiated dendritic cell, diffDC), which made the DC are not mature view of terminally differentiated cells, the antigen presentation after the completion of the task, as the micro environment in secondary lymphoid organs by, still can further proliferation and differentiation, which was given new life and function in different physiological or pathological conditions, diffDC interacts with each other around a lot of immune cells, the precise regulation for IL-10 secreted by activated NK cells and promote its ability to kill, and A selective chemoattractant IP-10 secretion of Thl cells and inhibit its proliferation, thereby maintaining the homeostasis. However, these views are from the experimental results in mice, the micro environment is how the role of the dendritic cells and splenic stroma in the human body will produce mature? Like mice like diffDC with negative regulation the function of human dendritic cell subsets? If there is such a DC subgroup, and what is the biological significance and the related mechanism?
In view of this topic is the interaction of the two kinds of cells, in this study we divided into three parts, respectively set up around the splenic stromal cells, the new regulation of human authentication three aspects the features and functions of dendritic cell subsets and cell subsets in the human body.
Establishment and identification of human spleen stromal cell line HESSCs
First, we isolated 4-6 month fetus fresh spleen, splenic stromal cells cultured by tissue. About a month or so, with stromal cells adherent culture plates were digested after the formation of splenic stromal cells. The initial splenic stromal cells that can be observed in a form of composition cell sizes, purity poor. With further training, gradually purified to stromal cells mainly by endothelial cell composition, cell growth rate tends to be stable, so after about 2-3 months of training, the formation of splenic stromal cell line stably.
Next, we conducted a preliminary identification of the human splenic stromal cells by flow cytometry analysis showed that the cell line is mainly composed of CD105+ endothelial cells, the expression of CD11c, CDllb and other myeloid cell marker.MRNA analysis showed that the cells within the skin type stromal cell line, detect detection by ELISA secretion the higher the TGF-p cell lines, suggesting that the cells may have certain differentiation ability, so we construct the splenic stromal cells is mainly composed of a group of endothelial cells composed of stromal cells with potential differentiation ability, which we call HESSCs (Human Endothelial-like Spleen Stromal Cells)
Two, the characteristics and functions of a new human regulated dendritic cell subgroup induced by HESSCs
In this part of the experiment, we focused on the characteristics and functions of CD11bhiCTLA-4+ novel human regulatory dendritic cell subsets differentiated from human peripheral blood monocyte derived mature dendritic cells induced by human spleen HESSCs.
First, we isolated human peripheral blood mononuclear cells from normal human peripheral blood samples (PBMC), for further purification of mononuclear cells of CD14+, GM-CSF and IL-4 combined with differentiation into immature dendritic cells (imDC), and the typical TLR ligand LPS to stimulate the differentiation of mature dendritic cells (maDC) after splenic stromal cells, the maDC and HESSC mixed culture will be built before, to investigate the interaction between the two. It was found that the removal of induced cytokines GM-CSF and IL-4, most of the control in 1-2 weeks of apoptosis or death of maDC medium, and most mixed with HESSCs cultured dendritic cells are still alive. That HESSCs supports the survival of monocyte derived maDC.
Based on the above phenomenon, we selected were co cultured with HESSC 1-2 weeks after maDC as the research object, detect the phenotype and function. The flow cytometry analysis showed that these cells relative to maDC mainly for CD11clow, CD11bhi, HLA-DRlow, CD80low, CD83low, CD86low and CTLA-4+, and cytokine assay confirmed that the cells secrete PGE2. TGF-p, however, has not yet found the group of cells can secrete higher IL-10, NO and other negative regulatory factor. Interestingly, the expression of inhibitory receptor CTLA-4, and it can produce high levels of PGE2, TGF-p and other negative regulation of cytokines, suggesting that the new group of dendritic cell subsets may some play immunesuppression. According to the analysis and comparison of mRNA maDC and the new type of dendritic cell subsets, verify the group TGF- beta model of dendritic cell subsets, the expression of mRNA in water CTLA-4 The level of IL-10, PD-1, PDL-1, PDL-2 and other important molecules of the dendritic cell subgroup were significantly higher than that of maDC.
Dendritic cells are the most important antigen-presenting cells, play an important role in the development of inflammation, so our group of new dendritic cell subsets in the inflammatory state and how to respond? Experiments show that the model of dendritic cell subsets compared with maDC in TLR4 ligand after LPS stimulation that inflammatory cytokines can secrete high levels of IL-10, TNF- alpha, PGE2 beta, TGF-. This result suggests that the new type of dendritic cell subsets in inflammatory conditions may play immunomodulatory effects even more.
HESSCs induced by this group of new regulatory dendritic cells and the expression of CTLA-4, CTLA-4 as an important negative regulator, play an important role in the inhibition of T cells during the immune response, so we speculate that the new type of dendritic cell subsets may have immunosuppressive function. Mixed lymphocyte reaction by in vitro experiments, we found that the new DC subsets can indeed significantly inhibited maDC stimulated allogeneic CD4+T cell proliferation, the specific mechanism needs further study.
As everyone knows, DC has a strong ability of phagocytosis in the immature state, when they encounter stimulation or differentiation after phagocytosis of phagocytic antigen basically lost. The phagocytic capacity of the new DC subsets by mature DC differentiation and is greatly enhanced, provide a great imagination on this phenomenon, we discuss deeply the study of its function.
In conclusion through the above experiment, found the maDC differentiation of splenic stromal cells HESSCs induced peripheral blood mononuclear cells from a high PGE2 people have a unique phenotype of CD1 model, 1bhiCTLA-4+ TGF- beta regulatory factor regulatory dendritic cell subsets. We named it CD11bhiCTLA-4+DCreg.
Three, identification of a new regulatory dendritic cell subgroup in the human body
In this part, we focus on finding and preliminarily verifying the existence of this new type of regulatory dendritic cells in the human spleen.
In order to verify the new DC subsets really exist in the body, we are looking for the people in the spleen of DC subgroup. We first used the new DC subgroup specific surface markers (CD11c+CD11bhiCTLA-4+) will be the same phenotype of cell sorting out, through the detection of cytokine secretion and inhibit its function to judge the human body if there is such a group of people have not been found in DC subsets.
We use the flow cytometry instrument of human spleen CD11c+ cell sorting for CD11chiHLA-DRhiCD11blow, CD11chiHLA-DRhiCD11bhi and CD11cintHLA-DRlowCD11bhi three cell subsets of the cell phenotype, mixed lymphocyte reaction, cytokine secretion and phagocytosis were detected compared to many aspects, confirmed the expression of CTLA-4 CD11chiHLA-DRhiCD11bhi memory in a group of human spleen cells, and secretion a large number of PGE2, TGF- beta, IL-10 and other cytokines, has strong phagocytic ability, can effectively inhibit the in vitro induced by maDC in a larger proportion of the cases of allogeneic CD4+T lymphocytes proliferation. So we determine the initial human spleen CD11chiHLA-DRhiCD11bhi cells in vitro and splenic stromal cell line HESSCs induced differentiation of CD11bhiCTLA-4+DCreg with features and functions similar.
In summary, we found through experiments, mature DC differentiation of human splenic stromal cells HESSCs induced human peripheral blood monocyte derived as a group has a negative regulatory role of immune model of human dendritic cell subsets. The cell subsets with CD11bhiCTLA-4+ cell phenotype, unique hypersecretion PGE2, a negative regulator of TGF- P factor, can effectively inhibit the proliferation of CD4+T lymphocytes, and verified the human (spleen) in existence. These results enrich the immune microenvironment to recognize negative regulation of immune response multicellular networks, for the further study of dendritic cells in the immune negative regulation mechanism provides a new direction and some the experimental basis for exploring tumor, provides a new way to study the self tolerance and autoimmune diseases such as the application of pathogenesis and immunotherapy.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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