嗜酸性粒细胞促进造血干细胞动员的调控机制研究

发布时间：2018-01-02 09:16

本文关键词：嗜酸性粒细胞促进造血干细胞动员的调控机制研究　出处：《浙江大学》2015年博士论文　论文类型：学位论文

【摘要】：背景支气管哮喘是一种由过敏原引起的由嗜酸性粒细胞(eosinophil, Eos),肥大细胞,T淋巴细胞等多种细胞和细胞组分参与的气道慢性炎症性疾病,其临床特点为气道高反应性(hyperresponsiveness, AHR),以及可逆性的气流受限。导致其病理的根本原因是Eos的气道局部的大量聚集,且Eos的数量与哮喘的严重程度相关。目前全球有3亿多人患有哮喘,中国哮喘患者已超过4000万。哮喘给家庭以及整个社会产生了严重的经济负担。目前以吸入型糖皮质激素为首选的治疗方案可以较好的控制哮喘的症状,但尚不能完全根治。目前对哮喘发病机制,病理学以及防治仍然是本领域研究的挑战,更好的研究Eos,并控制Eos为主的炎症也是本领域研究的热点。Eos是外周血的成熟白细胞的一种,呈椭圆形,因其胞浆内部充满粗大、整齐、均匀、排列紧密的嗜酸性颗粒而命名。正常人血液中嗜酸性粒细胞的水平较低,比例为白细胞总数的0.5%-5%,绝对值为0.05-0.5*10^9/L,Eos数量少但是作用非常重要。目前已经有很多关于Eos在哮喘中的病理机制研究,包括：聚集在气道周围的Eos释放其特异性的胞质颗粒蛋白如主要碱性蛋白-1 (major basic protein-1, MBP-1),嗜酸性粒细胞阳离子蛋白(eosinophil cationic protein, ECP)等破坏气道上皮细胞等局部组织；释放白介素-13 (interlukin, IL-13), IL-4等炎症因子,刺激杯状细胞增生,粘液高分泌以及AHR的产生；长期的慢性炎症过程中,Eos还分泌转化生长因子-β(Transforming growth factor-β, TGF-β)等与组织修复有关的蛋白,导致局部平滑肌增生,纤维沉积等病理现象。除参与哮喘的病理过程以外,Eos还在抗寄生虫感染的宿主免疫反应中其重要作用,包括分泌MBP直接破坏寄生虫细胞壁,分泌相应抗体而杀伤。Eos还是一种重要的免疫调节细胞,其功能包括激活肥大细胞,促进其分泌组胺和前列腺素等,参与后续的过敏性反应；以及处理并提呈一系列微生物,病毒和寄生虫抗原担任抗原提呈细胞的角色；Eos还能调节T淋巴细胞的功能,活化的Eos可以促进免疫反应中T细胞的增殖,并通过提呈可溶性抗原给CD4+T细胞来调节Th1/2的极化。Eos还参与肿瘤的免疫反应过程,如通过招募CD8+的T细胞来促进肿瘤细胞的杀伤作用等。Eos由造血干细胞在一系列复杂转录因子和细胞因子的调控之下定向分化而来,Eos是造血干细胞的终末成熟血细胞。二者之间除分化关系外并无其他研究报道。本团队前期研究发现在小鼠的哮喘模型中,出现骨髓与外周脾脏干细胞LSK(lineage-Sca-l+c-kit+)数量的增加,即干细胞的增殖和动员现象。而在Eos缺失的Phil小鼠中,则发现干细胞数量的减少。提示Eos可能反过来对其上级的造血干细胞有一种促进动员调控作用。基于此假设,我们利用了IL-5过表达导致的Eos大量存在的NJ.1638小鼠与Phil小鼠进行了一系列骨髓移植,体外共培养体系等实验,深入的研究了Eos与造血干细胞之间的调控关系。目的明确Eos对干细胞动员的调控作用,并进一步研究其机制。方法1.整体模型利用6-10周龄的同窝生的野生型(Wild Type, WT)小鼠与Phil小鼠,雌雄不限,以鸡卵清蛋白(OVA)致敏以及雾化攻击,建立经典的哮喘模型,以多色流式分析法标记骨髓与脾脏来源的LSK水平的干细胞,观察其增殖与动员的现象；分析NJ.1638体内的干细胞水平及其ROS水平,观察Eos大量增加对干细胞的影响；将NJ.1638与Phil小鼠杂交后分析骨髓与外周的干细胞水平,明确Eos对干细胞的调控关系；对WT和Phil小鼠腹腔注射经典的骨髓动员剂G-CSF,观察干细胞动员的现象。2.骨髓移植进行WT小鼠和NJ.1638小鼠之间的非竞争性和竞争性骨髓移植方案,观察Eos与干细胞活化的关系及其干细胞的分化功能；利用DCFH FA荧光染料标记活性氧自由基(ROS),检测ROS水平；利用流式分选的方法纯化LSK,与Eos进行体外培养,观察Eos对LSK的影响,找寻其机制。结果1.哮喘模型中,Eos的缺失导致干细胞动员现象的减弱。表现为与WT小鼠相比,Phil小鼠脾脏中干细胞数量增加减少。2.NJ.1638骨髓中LSK数量明显减少,而脾脏中LSK的数量则高于骨髓,提示干细胞动员的现象；NJ.1638体内的干细胞呈高ROS水平。3.NJ.1638与Phil小鼠杂交后体内高IL-5但缺乏Eos的小鼠中,干细胞动员的现象消失,提示Eos与干细胞动员直接相关。4.Eos不影响G-CSF导致的骨髓动员现象。5.NJ.1638与WT小鼠的骨髓移植实验说明Eos数量的变化与干细胞动员的趋势直接相关,且NJ.1638来源的干细胞分化能力下降。6.NJ.1638来源的干细胞归巢能力并没有下降。7.Eos与流式分选后的LSK共培养可以促进其ROS的产生。结论Eos数量的增加可以促进干细胞的动员,是通过增加干细胞的ROS水平导致的。
[Abstract]:Backgroundasthma is a caused by allergens by eosinophils (eosinophil, Eos), mast cells, T lymphocytes and other cells and cellular components involved in chronic inflammatory airway disease, the clinical features of airway hyperresponsiveness (hyperresponsiveness, AHR), and reversible airflow limitation at all. The cause of the pathology of Eos is the accumulation of local airway, and the severity of asthma and the number of the Eos. There are currently more than 300 million people worldwide suffer from asthma, Chinese asthma has more than 40 million. Asthma caused serious economic burden to the family and the society. At present, inhaled corticosteroids for the treatment of asthma control the preferred scheme can better the symptoms, but still can not completely cure. The pathogenesis of asthma, pathology and prevention is still the research challenges, research Eos better, A kind of mature white blood cells, inflammation is also the hotspot of.Eos research field control and Eos peripheral blood of the oval, because its internal cytoplasm is filled with thick, neat, uniform, closely spaced eosinophilic granules named. Eosinophils in blood of healthy people in the low level, the proportion of the total number of white blood cells 0.5%-5%, the absolute value of 0.05-0.5*10^9/L, the number of Eos is low but the effect is very important. At present, there has been a lot of study on pathological mechanism of Eos in asthma, including: the aggregation of cytoplasmic granule protein release and its specificity in the airway of Eos such as major basic protein -1 (major basic protein-1, MBP-1), eosinophil eosinophil cationic protein (eosinophil cationic, protein, ECP) of local tissue damage of airway epithelial cells; release of interleukin -13 (Interlukin, IL-13), IL-4 and other inflammatory cytokines, stimulated proliferation of goblet cells, mucus The production of AHR and hypersecretion; chronic inflammation in the process of long-term, Eos secretion of transforming growth factor beta (Transforming beta growth factor-, TGF-) and tissue repair related protein, leading to local smooth muscle hyperplasia, fibrous deposition and other pathological phenomena. In addition to the pathological process in asthma, Eos is still the host immune response against parasites infection plays a role, including the secretion of MBP parasites directly destroy the cell wall, and the secretion of antibodies against.Eos is a kind of important immune cells, its function include the activation of mast cells, promote the secretion of histamine and prostaglandins, involved in allergic reactions and subsequent treatment; and presenting a series of microorganism, virus and parasite antigen as antigen-presenting cells of the role; Eos can regulate the function of T lymphocytes, activated Eos can promote immune response in T cell proliferation, and The immune response by polarized.Eos presenting soluble antigen to CD4+T cells to regulate Th1/2 is also involved in cancer, such as through the recruitment of CD8+ T cells to promote tumor cell killing effect of.Eos hematopoietic stem cells in the regulation by a complex series of transcription factors and cytokines of differentiation to hematopoietic stem Eos the terminal cells of mature blood cells. In addition to the differentiation between the two relations not reported in other studies. The former research team found that in the asthma model mice, bone marrow and peripheral stem cells of spleen LSK (lineage-Sca-l+c-kit+) the increase in the number of stem cell mobilization and proliferation phenomenon. While in Eos deficient Phil mice it is found that, the number of stem cells is decreased. Suggesting that Eos may turn to its superior hematopoietic stem cells have a regulatory role to promote mobilization. Based on this hypothesis, we use the over expression of IL-5 induced The NJ.1638 mice and Phil mice Eos there are a lot of a series of bone marrow transplantation, the in vitro co culture system, in-depth study of the Eos and the relationship between hematopoietic stem cell regulation. Objective to make clear the regulation role of Eos on stem cell mobilization, and study its mechanism. Methods 1. week old model using 6-10 the wild type littermates (Wild Type, WT) and Phil mice, male and female, with chicken ovalbumin (OVA) sensitization and atomization attack, the asthma model was established by classical, multi-color flow LSK horizontal analysis method and labeled bone marrow spleen derived stem cells, to observe the proliferation and mobilization of the phenomenon analysis; NJ.1638 in vivo stem cell level and the level of ROS, Eos observed a substantial increase in the stem cells; will be hybrid NJ.1638 and Phil mice after analysis of stem cell of bone marrow and peripheral, clear Eos of stem cell regulation relationship The classic WT and intraperitoneal injection; Phil mice bone marrow mobilization agent G-CSF, non competitive and competitive bone marrow transplantation to observe stem cell mobilization of bone marrow transplantation of.2. between WT and NJ.1638 mice, and observe the Eos stem cell activation and differentiation of stem cells; using DCFH FA fluorescent dye labeled activity oxygen free radical (ROS), to detect the level of ROS; using the method of flow separation and purification of LSK and Eos, were cultured in vitro and the effect of Eos on LSK, to find the mechanism. Results of the 1. models of asthma, the loss of Eos causes stem cells weakened mobilization phenomenon. As compared with WT mice, stem cells the number increased to reduce the amount of.2.NJ.1638 in bone marrow LSK significantly reduced spleen of Phil mice, while the number of spleen in LSK is higher than that of bone marrow stem cell mobilization, suggesting that the phenomenon of NJ.1638 in vivo; stem cells showed high levels of.3.NJ.1638 and P ROS HIL mice after hybridization in high IL-5 but Eos deficient mice, disappearance of stem cell mobilization, suggesting that Eos and stem cell mobilization is directly related to.4.Eos does not affect the G-CSF induced bone marrow mobilization phenomenon of.5.NJ.1638 and WT mice bone marrow transplantation experiment shows that the number of Eos is directly related to stem cell mobilization, stem cell differentiation and the source of NJ.1638 decreased stem cell homing ability of.6.NJ.1638 derived.7.Eos did not decline with flow cytometry after LSK co culture can promote the production of ROS increased. Conclusion the number of Eos can promote stem cell mobilization, by increasing the level of ROS in stem cells.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R562.25

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