本文關(guān)鍵詞：嗜酸性粒細(xì)胞促進(jìn)造血干細(xì)胞動(dòng)員的調(diào)控機(jī)制研究　出處：《浙江大學(xué)》2015年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： Eos 哮喘 干細(xì)胞 動(dòng)員 ROS

【摘要】：背景支氣管哮喘是一種由過(guò)敏原引起的由嗜酸性粒細(xì)胞(eosinophil, Eos),肥大細(xì)胞,T淋巴細(xì)胞等多種細(xì)胞和細(xì)胞組分參與的氣道慢性炎癥性疾病,其臨床特點(diǎn)為氣道高反應(yīng)性(hyperresponsiveness, AHR),以及可逆性的氣流受限。導(dǎo)致其病理的根本原因是Eos的氣道局部的大量聚集,且Eos的數(shù)量與哮喘的嚴(yán)重程度相關(guān)。目前全球有3億多人患有哮喘,中國(guó)哮喘患者已超過(guò)4000萬(wàn)。哮喘給家庭以及整個(gè)社會(huì)產(chǎn)生了嚴(yán)重的經(jīng)濟(jì)負(fù)擔(dān)。目前以吸入型糖皮質(zhì)激素為首選的治療方案可以較好的控制哮喘的癥狀,但尚不能完全根治。目前對(duì)哮喘發(fā)病機(jī)制,病理學(xué)以及防治仍然是本領(lǐng)域研究的挑戰(zhàn),更好的研究Eos,并控制Eos為主的炎癥也是本領(lǐng)域研究的熱點(diǎn)。Eos是外周血的成熟白細(xì)胞的一種,呈橢圓形,因其胞漿內(nèi)部充滿粗大、整齊、均勻、排列緊密的嗜酸性顆粒而命名。正常人血液中嗜酸性粒細(xì)胞的水平較低,比例為白細(xì)胞總數(shù)的0.5%-5%,絕對(duì)值為0.05-0.5*10^9/L,Eos數(shù)量少但是作用非常重要。目前已經(jīng)有很多關(guān)于Eos在哮喘中的病理機(jī)制研究,包括：聚集在氣道周圍的Eos釋放其特異性的胞質(zhì)顆粒蛋白如主要堿性蛋白-1 (major basic protein-1, MBP-1),嗜酸性粒細(xì)胞陽(yáng)離子蛋白(eosinophil cationic protein, ECP)等破壞氣道上皮細(xì)胞等局部組織；釋放白介素-13 (interlukin, IL-13), IL-4等炎癥因子,刺激杯狀細(xì)胞增生,粘液高分泌以及AHR的產(chǎn)生；長(zhǎng)期的慢性炎癥過(guò)程中,Eos還分泌轉(zhuǎn)化生長(zhǎng)因子-β(Transforming growth factor-β, TGF-β)等與組織修復(fù)有關(guān)的蛋白,導(dǎo)致局部平滑肌增生,纖維沉積等病理現(xiàn)象。除參與哮喘的病理過(guò)程以外,Eos還在抗寄生蟲感染的宿主免疫反應(yīng)中其重要作用,包括分泌MBP直接破壞寄生蟲細(xì)胞壁,分泌相應(yīng)抗體而殺傷。Eos還是一種重要的免疫調(diào)節(jié)細(xì)胞,其功能包括激活肥大細(xì)胞,促進(jìn)其分泌組胺和前列腺素等,參與后續(xù)的過(guò)敏性反應(yīng)；以及處理并提呈一系列微生物,病毒和寄生蟲抗原擔(dān)任抗原提呈細(xì)胞的角色；Eos還能調(diào)節(jié)T淋巴細(xì)胞的功能,活化的Eos可以促進(jìn)免疫反應(yīng)中T細(xì)胞的增殖,并通過(guò)提呈可溶性抗原給CD4+T細(xì)胞來(lái)調(diào)節(jié)Th1/2的極化。Eos還參與腫瘤的免疫反應(yīng)過(guò)程,如通過(guò)招募CD8+的T細(xì)胞來(lái)促進(jìn)腫瘤細(xì)胞的殺傷作用等。Eos由造血干細(xì)胞在一系列復(fù)雜轉(zhuǎn)錄因子和細(xì)胞因子的調(diào)控之下定向分化而來(lái),Eos是造血干細(xì)胞的終末成熟血細(xì)胞。二者之間除分化關(guān)系外并無(wú)其他研究報(bào)道。本團(tuán)隊(duì)前期研究發(fā)現(xiàn)在小鼠的哮喘模型中,出現(xiàn)骨髓與外周脾臟干細(xì)胞LSK(lineage-Sca-l+c-kit+)數(shù)量的增加,即干細(xì)胞的增殖和動(dòng)員現(xiàn)象。而在Eos缺失的Phil小鼠中,則發(fā)現(xiàn)干細(xì)胞數(shù)量的減少。提示Eos可能反過(guò)來(lái)對(duì)其上級(jí)的造血干細(xì)胞有一種促進(jìn)動(dòng)員調(diào)控作用�；诖思僭O(shè),我們利用了IL-5過(guò)表達(dá)導(dǎo)致的Eos大量存在的NJ.1638小鼠與Phil小鼠進(jìn)行了一系列骨髓移植,體外共培養(yǎng)體系等實(shí)驗(yàn),深入的研究了Eos與造血干細(xì)胞之間的調(diào)控關(guān)系。目的明確Eos對(duì)干細(xì)胞動(dòng)員的調(diào)控作用,并進(jìn)一步研究其機(jī)制。方法1.整體模型利用6-10周齡的同窩生的野生型(Wild Type, WT)小鼠與Phil小鼠,雌雄不限,以雞卵清蛋白(OVA)致敏以及霧化攻擊,建立經(jīng)典的哮喘模型,以多色流式分析法標(biāo)記骨髓與脾臟來(lái)源的LSK水平的干細(xì)胞,觀察其增殖與動(dòng)員的現(xiàn)象；分析NJ.1638體內(nèi)的干細(xì)胞水平及其ROS水平,觀察Eos大量增加對(duì)干細(xì)胞的影響；將NJ.1638與Phil小鼠雜交后分析骨髓與外周的干細(xì)胞水平,明確Eos對(duì)干細(xì)胞的調(diào)控關(guān)系；對(duì)WT和Phil小鼠腹腔注射經(jīng)典的骨髓動(dòng)員劑G-CSF,觀察干細(xì)胞動(dòng)員的現(xiàn)象。2.骨髓移植進(jìn)行WT小鼠和NJ.1638小鼠之間的非競(jìng)爭(zhēng)性和競(jìng)爭(zhēng)性骨髓移植方案,觀察Eos與干細(xì)胞活化的關(guān)系及其干細(xì)胞的分化功能；利用DCFH FA熒光染料標(biāo)記活性氧自由基(ROS),檢測(cè)ROS水平；利用流式分選的方法純化LSK,與Eos進(jìn)行體外培養(yǎng),觀察Eos對(duì)LSK的影響,找尋其機(jī)制。結(jié)果1.哮喘模型中,Eos的缺失導(dǎo)致干細(xì)胞動(dòng)員現(xiàn)象的減弱。表現(xiàn)為與WT小鼠相比,Phil小鼠脾臟中干細(xì)胞數(shù)量增加減少。2.NJ.1638骨髓中LSK數(shù)量明顯減少,而脾臟中LSK的數(shù)量則高于骨髓,提示干細(xì)胞動(dòng)員的現(xiàn)象；NJ.1638體內(nèi)的干細(xì)胞呈高ROS水平。3.NJ.1638與Phil小鼠雜交后體內(nèi)高IL-5但缺乏Eos的小鼠中,干細(xì)胞動(dòng)員的現(xiàn)象消失,提示Eos與干細(xì)胞動(dòng)員直接相關(guān)。4.Eos不影響G-CSF導(dǎo)致的骨髓動(dòng)員現(xiàn)象。5.NJ.1638與WT小鼠的骨髓移植實(shí)驗(yàn)說(shuō)明Eos數(shù)量的變化與干細(xì)胞動(dòng)員的趨勢(shì)直接相關(guān),且NJ.1638來(lái)源的干細(xì)胞分化能力下降。6.NJ.1638來(lái)源的干細(xì)胞歸巢能力并沒有下降。7.Eos與流式分選后的LSK共培養(yǎng)可以促進(jìn)其ROS的產(chǎn)生。結(jié)論Eos數(shù)量的增加可以促進(jìn)干細(xì)胞的動(dòng)員,是通過(guò)增加干細(xì)胞的ROS水平導(dǎo)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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R562.25

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