雷帕霉素与细胞自噬在哮喘气道炎症和嗜酸粒细胞分化中的作用研究

发布时间：2018-05-11 13:51

本文选题：雷帕霉素 + 细胞自噬　；参考：《浙江大学》2013年博士论文

【摘要】：支气管哮喘(简称哮喘)是一种以嗜酸性粒细胞(Eosinophils, Eos)、肥大细胞和T淋巴细胞浸润为主的气道慢性炎症性疾病,表现为气道高反应性和可逆性气流受限。其中嗜酸性粒细胞是哮喘过敏性气道炎症反应的主要效应细胞,Eos与哮喘发病之间存在直接因果关系。研究发现,嗜酸性粒细胞在哮喘发病过程中参与多种功能调控,包括抗原提呈,细胞因子、趋化因子、颗粒介质以及白三烯的释放等。嗜酸性粒细胞是由骨髓共同髓系祖细胞(CMP)经由粒-单核系祖细胞(GMP),由嗜酸性粒细胞祖细胞(EoP)定向分化成熟。嗜酸性粒细胞分化涉及多种转录因子(GATA-1等)和细胞因子,如白介素3(IL-3),IL-5,粒-单核集落刺激因子(GM-CSF),其中IL-5对Eos最终分化成熟起最主要的调节作用。然而,目前对于哮喘发病过程中Eos调控机制的研究并不多. 细胞自噬(autophagy)是机体一种重要的防御和保护机制。在某些特定的微环境条件(如饥饿,生长素缺乏)下,细胞内形成一种双层膜结构的囊泡,细胞自噬体(autophagosomes),并同时捕获细胞内的受损的细胞器(如线粒体,内质网)和变性的蛋白质等；然后该囊泡与溶酶体溶合,形成自噬溶酶体(autolysosomes)。在溶酶体水解酶等的作用下,自噬体所包含的各种细胞器和生物大分子得以消化和降解,从而为重新合成新的生物大分子提供原料和能量。因此从本义上讲,细胞自噬是机体保持内稳态(homeostasis)和适应微环境改变的一种重要的自我调节和保护机制。然而,在某些特定的条件下,细胞器和各种生物大分子的过量消耗最终会导致细胞的另一种程序性死亡,细胞自噬死亡(autophagic cell death)。由于细胞自噬能保护或者促进细胞死亡,因此在不同的疾病病变过程中,其功能截然不同。越来越多的研究表明,自噬在机体的免疫、感染、炎症、肿瘤、心血管病、神经退行性病的发病中具有十分重要的作用。然而,细胞自噬在呼吸系统的研究并不多。新近有研究开创性地阐明了细胞自噬在吸烟诱导的气道上皮细胞损伤以及慢性阻塞性肺疾病(COPD)形成过程中的重要调控作用。然而,细胞自噬在支气管哮喘分子发病机制中的作用鲜有研究报导。雷帕霉素(rapamycin)是经典的细胞自噬诱导剂,是1975年从加拿大Easter岛上的吸水链霉菌中提取的一种大环内酯类抗生素。雷帕霉素的主要作用靶点,哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)能接受并整合生长因子、能量、氧气和氨基酸四大主要信号,参与调节能量代谢、蛋白质、脂质、细胞器的合成以及细胞自噬等生理过程,在细胞的生长、增殖、分化和凋亡中起着重要的调控作用,从而维持机体与细胞的稳态平衡。研究发现雷帕霉素在哮喘动物模型中的作用并不一致,有报道发现雷帕霉素能抑制过敏性气道炎症,气道高反应性,杯状细胞化生和IgE产生,但也有部分研究认为雷帕霉素对过敏性气道炎症和气道高反应性没有影响。然而,最关键的是这些研究均只简单利用雷帕霉素体内干预观察哮喘表型,并未阐明雷帕霉素及mTOR在哮喘发病机制中起到关键调控作用及其具体机制。越来越多的研究发现细胞自噬和mTOR在造血干细胞分化和增殖中起到重要作用。自噬缺陷可抑制红系造血导致严重贫血、抑制T淋巴细胞、B淋巴细胞数目和功能,而mTOR通过调控T细胞分化、功能、代谢参与调控适应性免疫应答。由此,我们假设细胞自噬和mTOR也参与骨髓粒系祖细胞尤其是嗜酸性粒细胞分化的调控,并进一步探讨其在以嗜酸性粒细胞气道炎症为主要表现的整体哮喘模型中的可能作用与贡献,从而为哮喘分子发病机制的研究开拓新的视野,为哮喘临床的防治提供新的靶点。本实验分两部分进行研究：(1)探求雷帕霉素及mTOR在哮喘气道炎症和骨髓嗜酸性粒细胞分化中的作用；(2)利用细胞自噬相关基因敲除小鼠Beclin1+/-和骨髓移植技术,分别阐明细胞自噬在气道上皮细胞损伤和骨髓嗜酸性粒细胞分化中的不同调控作用。第一部分雷帕霉素在哮喘气道炎症和嗜酸粒细胞分化中的作用研究目的：研究雷帕霉素干预对哮喘气道炎症的影响,并探讨雷帕霉素在OVA诱导肺组织T细胞免疫应答及骨髓嗜酸性粒细胞分化过程中的调控作用。方法：健康雌性C57BL/6小鼠,随机分为四组：生理盐水对照组(SHAM组)、模型组(OVA组)、雷帕霉素对照组(SHAM+RAPA组)、雷帕霉素哮喘组(OVA+RAPA组)。以卵白蛋白(OVA)致敏和激发建立哮喘模型,生理盐水对照组以同等剂量的生理盐水代替OVA,雷帕霉素组在每次抗原激发前1小时进行腹腔注射(1mg/kg)。于最后一次抗原激发后24小时检测肺泡灌洗液(BALF)、外周血和骨髓中的嗜酸性粒细胞数；肺组织病理切片观察气道炎症细胞浸润情况,ELISA检测血清中IL-5,IL-13水平。流式细胞术检测和分析肺组织Th2, Th17, Treg各T细胞亚群的比例,同时检测骨髓中嗜酸性粒细胞祖细胞(EoP, Lin-Sca-l-CD34+IL-5Ra+c-Kitl0)的比例。Western blot检测骨髓嗜酸性粒细胞分化过程中mTOR水平变化,体外克隆形成试验及骨髓Eos诱导分化培养检测雷帕霉素干预对骨髓嗜酸性粒细胞分化及功能的影响。最后利用IL-5转基因小鼠NJ.1638,连续3天腹腔注射雷帕霉素,检测外周血和骨髓中嗜酸性粒细胞数,ELISA检测血清中IL-5水平,流式细胞术检测骨髓EoP的比例以及嗜酸性粒细胞凋亡情况。结果：雷帕霉素干预后显著缓解OVA诱导的气道过敏性炎症,但是不影响肺组织中Th2细胞,Th17细胞和Treg水平。雷帕霉素抑制了气道局部以及外周血,骨髓嗜酸性粒细胞数目,但是并不改变血清IL-5水平。体外克隆形成试验和骨髓Eos诱导分化培养发现,雷帕霉素可以直接抑制IL-5介导的嗜酸性粒细胞分化及培养上清Eos分泌IL-6和IL-13的水平。离体骨髓克隆形成试验及骨髓流式细胞术检测结果提示,雷帕霉素对嗜酸性粒细胞分化的抑制最终导致骨髓嗜酸性粒细胞祖细胞的聚集。对IL-5转基因小鼠NJ.1638的研究同样证实了雷帕霉素对外周血和骨髓嗜酸性粒细胞数的抑制作用及骨髓EoP的聚集,并不依赖其血清高表达IL-5水平的改变,也不影响嗜酸性粒细胞的凋亡。结论：雷帕霉素可以缓解哮喘小鼠气道过敏性炎症,可能是通过抑制骨髓嗜酸性粒细胞分化及功能,而对Eos凋亡和肺组织T细胞免疫无明显影响。第二部分细胞自噬在哮喘气道炎症和嗜酸粒细胞分化中的作用研究目的：研究细胞自噬敲除对哮喘整体模型的影响,并探讨细胞自噬在OVA诱导的气道上皮细胞损伤和骨髓嗜酸性粒细胞分化过程中的不同调控作用。方法：通过Western blot检测哮喘小鼠骨髓及体外嗜酸性粒细胞诱导分化过程中自噬水平变化,并利用细胞自噬相关基因缺陷小鼠Beclin1+/-,通过瑞士-吉姆萨染色分析其外周血和骨髓中嗜酸性粒细胞的水平,流式细胞术检测其骨髓嗜酸性粒细胞祖细胞水平,并通过甲基纤维素克隆形成试验检测其骨髓嗜酸性粒细胞分化能力。同时利用Beclin1+/-小鼠及其同窝生WT小鼠以OVA致敏和激发建立哮喘模型,随机分组如下：野生型生理盐水对照组(WT/NS),野生型哮喘模型组(WT/OVA),Beclin1+/-小鼠生理盐水对照组(BECN/NS)和Beclin1+/-小鼠哮喘模型组(BECN/OVA)。末次激发后24小时,采用有创方法测定小鼠气道反应性,并检测气道灌洗液中的白细胞总数和嗜酸性粒细胞数；肺组织病理切片观察气道炎症细胞浸润和粘液分泌情况,电镜观察哮喘小鼠气道上皮细胞自噬泡聚集情况。体外培养人正常肺上皮细胞(BEAS-2B),Western blot检测OVA体外干预过程中自噬水平变化,Q-PCR检测饥饿诱导自噬及IL-13体外干预对粘蛋白MUC5AC mRNA的表达情况。最后采用骨髓移植的方法,白硝胺+环磷酰胺联合化疗毁损野生型小鼠骨髓造血系统,然后将Beclin1+/-小鼠或同窝生野生型小鼠骨髓移植至野生型小鼠并重建其造血系统,OVA诱导建立哮喘模型,根据回输骨髓不同分两组：WT-WT-OVA组和BECN-WT-OVA组,观察骨髓局部自噬敲除对哮喘整体模型气道高反应和气道炎症的贡献。结果：细胞自噬相关基因敲除小鼠Beclin1+/-,与同窝生野生型(WT)小鼠相比,外周血和骨髓中嗜酸性粒细胞数目显著增多,骨髓EoP水平虽有下降趋势,但无统计学意义。体外Eos诱导分化过程中LC3B蛋白水平的改变提示细胞自噬参与骨髓嗜酸性粒细胞分化。体外克隆形成试验直接证实,Beclin1+/-小鼠骨髓生成嗜酸性粒细胞克隆形成单位能力增加。但Beclin1+/-小鼠抗原激发后气道反应性、气道炎症、粘液高分泌,与野生型小鼠模型组相比并未恶化,反而出现明显减轻。同时发现,虽然哮喘小鼠骨髓中LC3B蛋白水平下降,但电镜可见哮喘气道上皮细胞中存在细胞自噬泡聚集。并且体外实验表明,OVA干预可上调LC3B蛋白水平,饥饿诱导自噬可进一步增强IL-13介导的BEAS-2B细胞MUC5AC mRNA的表达。在骨髓移植实验中,Beclin1+/"小鼠骨髓回输的哮喘小鼠(BECN-WT-OVA组),气道反应性和气道炎症都明显高于野生型小鼠骨髓回输的哮喘小鼠(WT-WT-OVA组)。结论：细胞自噬敲除虽然在动物模型中整体表现为可以缓解哮嗤小鼠气道过敏性炎症,气道高反应性和粘液高分泌,但细胞自噬在气道局部损伤和骨髓Eos分化过程中可能存在两种完全相反的调控机制。自噬敲除抑制了OVA诱导的气道上皮细胞损伤从而缓解了哮喘表型,而在骨髓嗜酸性粒细胞分化过程中则促进了其分化能力从而恶化了哮喘气道炎症。我们的研究提示对细胞自噬和mTOR信号通路的研究有望为哮喘的防治提供新的靶点。
[Abstract]:Bronchial asthma (asthma) is a chronic airway inflammatory disease characterized by eosinophil (Eosinophils, Eos), mast cell and T lymphocyte infiltration, characterized by airway hyperresponsiveness and reversible airflow limitation. Eosinophils are the main effector cells of asthma allergic airway inflammation, Eos and asthma There is a direct causal relationship between diseases. The study found that eosinophils participate in a variety of functional regulation during the pathogenesis of asthma, including antigen presentation, cytokine, chemokines, granular media and release of leukotrienes. Eosinophils are derived from bone marrow common myeloid progenitor cells (CMP) via granulocyte mononuclear progenitor cells (GMP), by eosinophilic acid. Granulocyte progenitor cells (EoP) are differentiated and mature. Eosinophil differentiation involves a variety of transcription factors (GATA-1) and cytokines, such as interleukin 3 (IL-3), IL-5, and granulocyte colony stimulating factor (GM-CSF), of which IL-5 plays the most important role in the final differentiation and maturation of Eos. However, at present, the regulation mechanism of Eos in the pathogenesis of asthma is present. There is not much research.
Cellular autophagy (autophagy) is an important mechanism for the defense and protection of the body. In certain specific microenvironmental conditions (such as starvation, auxin deficiency), the cell forms a double layer membrane vesicle, the cell autophago (autophagosomes), and simultaneously captures the damaged organelles (such as mitochondria, endoplasmic reticulum) and denatured proteins in the cells. In addition, the vesicles dissolve with lysosomes to form autophagic lysosomes (autolysosomes). Under the action of lysosomal hydrolase, the various organelles and biological macromolecules contained in the autophagosome can be digested and degraded to provide raw materials and energy for the resynthesis of new biological macromolecules. Therefore, autophagy is the machine. An important self-regulation and protection mechanism for maintaining internal homeostasis (homeostasis) and adapting to microenvironment changes. However, under certain conditions, excessive consumption of organelles and various biological macromolecules will eventually lead to another programmed cell death, cell autophagy death (autophagic cell death). A growing number of studies have shown that autophagy plays a very important role in the pathogenesis of immune, infection, inflammation, cancer, cardiovascular disease, and neurodegenerative disease. However, there are few studies on the cell autophagy in the respiratory system. The study elucidates the important regulatory role of autophagy in smoking induced airway epithelial cell injury and the formation of chronic obstructive pulmonary disease (COPD). However, the role of autophagy in the molecular pathogenesis of bronchial asthma is rarely reported.
Rapamycin (rapamycin) is a classic cell autophagy inducer, a macrolide antibiotic extracted from Streptomyces bibula on the Canadian Easter island in 1975. The main target of rapamycin, the mammalian mammalian target of rapamycin, mTOR, can accept and integrate growth factors, energy, and oxygen. The four major signals of gas and amino acids are involved in regulating energy metabolism, protein, lipid, synthesis of organelles and cell autophagy, which play an important regulatory role in cell growth, proliferation, differentiation and apoptosis, thus maintaining the homeostasis of the body and cells. The study of rapamycin in the animal model of asthma has been found. It is not consistent with reports that rapamycin can inhibit allergic airway inflammation, airway hyperresponsiveness, goblet cell production and IgE production, but some studies have also found that rapamycin has no effect on allergic airway inflammation and airway hyperresponsiveness. However, the key is that these studies only simply use rapamycin in vivo intervention. The observation of asthma phenotype did not clarify the key regulatory role of rapamycin and mTOR in the pathogenesis of asthma and its specific mechanism.
More and more studies have found that autophagy and mTOR play an important role in the differentiation and proliferation of hematopoietic stem cells. Autophagy can inhibit red blood hematopoiesis to cause severe anemia, inhibit the number and function of T lymphocytes, B lymphocytes, and mTOR by regulating T cell differentiation, function, metabolite and adaptive immune response. Cellular autophagy and mTOR are also involved in the regulation of bone marrow granulocyte progenitor cells, especially eosinophil differentiation, and further explore its possible role and contribution in the overall asthma model which is the main manifestation of eosinophil airway inflammation, thus developing a new field of vision for the study of the pathogenesis of asthma and preventing the clinical prevention of asthma. The treatment provides new targets.
This experiment is divided into two parts: (1) to explore the role of rapamycin and mTOR in airway inflammation and bone marrow eosinophil differentiation in asthma. (2) the use of Beclin1+/- and bone marrow transplantation in autophagy related gene knockout mice to elucidate cell autophagy in airway epithelial cell damage and bone marrow eosinophil differentiation, respectively. Different regulatory functions.
Part one effect of rapamycin on airway inflammation and eosinophil differentiation in asthmatic rats
Objective: To study the effect of rapamycin on airway inflammation in asthma and to explore the role of rapamycin in the regulation of T cell immune response and bone marrow eosinophil differentiation induced by OVA in lung tissue.
Methods: healthy female C57BL/6 mice were randomly divided into four groups: normal saline control group (group SHAM), model group (group OVA), rapamycin control group (group SHAM+RAPA), rapamycin asthma group (group OVA+RAPA). Asthma model was established with albumin (OVA) sensitization and stimulation, and saline control group replaced OVA with equal dose of saline in saline control group. Mycophenin group was injected intraperitoneally at 1 hours before each antigen excitation (1mg/kg). Pulmonary alveolar lavage fluid (BALF), eosinophil number in peripheral blood and bone marrow were detected at 24 hours after the last antigen excitation. The infiltration of airway inflammatory cells was observed by pathological sections of lung tissue, and IL-5 and IL-13 levels in serum were detected by ELISA. Flow cytometry And analyze the proportion of Th2, Th17, Treg T cell subsets in lung tissue, and detect the proportion of EoP (Lin-Sca-l-CD34+IL-5Ra+c-Kitl0) in bone marrow,.Western blot to detect the change of mTOR level during the differentiation of bone marrow eosinophils. In vitro cloning and formation test and bone marrow Eos induced differentiation and differentiation of rapamycin The effect of intervention on the differentiation and function of bone marrow eosinophils. Finally, using IL-5 transgenic mice NJ.1638, the number of eosinophils in peripheral blood and bone marrow was detected by intraperitoneal injection of rapamycin for 3 days. The level of IL-5 in serum was detected by ELISA, the proportion of EoP in bone marrow by flow cytometry and apoptosis of eosinophils were detected by flow cytometry.
Results: rapamycin induced OVA induced airway anaphylaxis significantly, but did not affect Th2 cells, Th17 cells and Treg levels in the lung tissue. Rapamycin inhibited the local and peripheral blood of the airway, the number of eosinophils in the bone marrow, but did not change the serum IL-5 level. In vitro cloning and formation test and bone marrow Eos induction. In vitro culture, rapamycin can directly inhibit the differentiation of IL-5 mediated eosinophils and the level of Eos secretion of IL-6 and IL-13 in supernatant. In vitro bone marrow cloning test and bone marrow flow cytometry results suggest that the inhibition of rapamycin on eosinophil differentiation eventually leads to bone marrow eosinophil progenitor cells The study of NJ.1638 in IL-5 transgenic mice also confirmed the inhibitory effect of rapamycin on the number of peripheral blood and bone marrow eosinophils and the aggregation of bone marrow EoP, which did not depend on the changes in the level of the serum high expression of IL-5, and did not affect the apoptosis of eosinophils.
Conclusion: rapamycin can alleviate the airway allergic inflammation in asthmatic mice, possibly by inhibiting the differentiation and function of bone marrow eosinophils, but has no obvious effect on the apoptosis of Eos and the immunity of T cells in the lung tissue.
The second part is the role of autophagy in airway inflammation and eosinophil differentiation in asthma.
Objective: To investigate the effect of autophagy knockout on the overall model of asthma, and to explore the different regulatory effects of autophagy on OVA induced airway epithelial cell damage and bone marrow eosinophil differentiation.
Methods: the changes of autophagy in the bone marrow and eosinophil induced differentiation of asthmatic mice were detected by Western blot, and the level of eosinophils in peripheral blood and bone marrow was analyzed by Swiss Giemsa staining, and the eosinophilia of bone marrow was detected by flow cytometry. The level of granulocyte progenitor cells and the ability to detect the differentiation of bone marrow eosinophils by the methyl cellulose clone formation test. At the same time, the asthma model was established by using Beclin1+/- mice and the same nest WT mice with OVA sensitization and stimulation. The random groups were as follows: the wild type physiological saline group (WT/NS), the wild type asthma model group (WT/OVA), Bec Lin1+/- mice in normal saline control group (BECN/NS) and Beclin1+/- mice asthma model group (BECN/OVA). 24 hours after the last stimulation, the airway responsiveness of mice was measured by invasive method, and the total number of leukocytes and eosinophils in the airway lavage fluid were detected. The infiltration of airway inflammatory cells and mucus secretion were observed in the lung tissue section. The aggregation of autophagic vesicles in airway epithelial cells of asthmatic mice was observed by electron microscopy. Normal lung epithelial cells (BEAS-2B) were cultured in vitro. Western blot was used to detect the change of autophagy during the intervention of OVA in vitro. Q-PCR was used to detect the expression of autophagy induced by starvation and IL-13 in vitro intervention on mucin MUC5AC mRNA. Finally, the method of bone marrow transplantation was used. The bone marrow hematopoietic system of wild type mice was damaged by combined chemotherapy of nitrosamines and cyclophosphamide, then the bone marrow of Beclin1+/- mice or the same fossa wild type mice was transplanted into the wild type mice and the hematopoietic system was rebuilt. The asthma model was induced by OVA, and the autophagy of the bone marrow was observed in two groups: group WT-WT-OVA and BECN-WT-OVA. Knockout contributes to airway hyperresponsiveness and airway inflammation in the overall asthma model.
Results: the number of eosinophils in peripheral blood and bone marrow was significantly increased and the level of bone marrow EoP decreased, but there was no significant difference in the number of eosinophils in the peripheral blood and bone marrow in mice with autophagy related gene knockout Beclin1+/- mice. The changes in the level of LC3B protein in the process of Eos induced differentiation in vitro suggest that the autophagy is involved in the bone marrow eosinophilia. In vitro clone formation test directly confirmed that the ability of Beclin1+/- mouse bone marrow eosinophil clone formation unit increased. However, the airway reactivity, airway inflammation and mucus hypersecretion of Beclin1+/- mice were not deteriorated compared with the wild type mouse model group. However, the LC3B protein level in the bone marrow of the asthmatic mice decreased, but the electron microscope showed that there was a cell autophagic vesicle aggregation in the bronchial epithelial cells of asthma. In vitro experiments showed that OVA intervention could increase the level of LC3B protein. The expression of MUC5AC mRNA in BEAS-2B cells mediated by IL-13 was further enhanced by starvation induced autophagy. In the bone marrow transplantation experiment, Beclin1+/ "is small. Airway inflammation and airway inflammation were significantly higher in asthmatic mice (BECN-WT-OVA group) than those in wild type mice (WT-WT-OVA group).
Conclusion: Although autophagy knockout in the animal model can alleviate airway allergic inflammation, airway hyperresponsiveness and mucus hypersecretion, there may be two completely opposite regulatory mechanisms in the process of airway local injury and bone marrow Eos differentiation. Autophagic knockout inhibits the OVA induced airway. Our study suggests that the study of autophagy and mTOR signaling pathways may provide a new approach to the prevention and treatment of asthma.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R562.25

【相似文献】