维生素D对中性粒细胞哮喘小鼠模型的炎症抑制作用及可能机制

发布时间：2018-05-18 13:30

  本文选题：支气管哮喘 + 中性粒细胞　； 参考：《南方医科大学》2013年博士论文

【摘要】：目前激素是治疗哮喘最有效的药物,但是仍然有部分哮喘患者尽管使用了大剂量的激素,却不能获得有效的控制。这部分哮喘称为激素抵抗型哮喘,有研究显示这部分病人诱导痰,以中性粒细胞为主。目前对于哮喘表型的研究是热点问题,因为以哮喘表型为基础对病人进行分类,能够使哮喘患者得到更加个性化的治疗。以诱导痰炎症细胞的种类对哮喘表型分类主要有一下四种类型：嗜酸性粒细胞型,中性粒细胞型,混合粒细胞型及少粒细胞型。目前对于为什么中性粒细胞哮喘对吸入激素抵抗原因还不清楚,中性粒细胞是激素抵抗的一个原因,还是说它只是激素抵抗哮喘患者的一个表现,还存在很大争议。初步的研究结果显示,激素能够抑制中性粒细胞凋亡可能是导致激素抵抗的一个原因。对于中性粒细胞哮喘表型的研究尚不深入,主要原因是没有可靠的动物模型,迄今为止哮喘动物模型最常用的致敏原为鸡卵清白蛋白(OVA),而OVA诱导的哮喘模型是以嗜酸性粒细胞为主,且应用OVA诱导哮喘100多年来,使得研究者对于哮喘的发病机制的认识有了很大的进步,药物的研发也获得一定程度的成功。因此我们试图建立一种稳定可靠的哮喘动物模型作为平台来研究中性粒细胞型哮喘。 甲苯二异氰酸酯(TDI),一种常用的工业原料,是成为职业性哮喘最为重要的诱因,但是随着我们生活方式的改变,这种主要用于油漆及泡沫材料中的工业原料,已经变得和我们每个人的生后都息息相关。临床研究发现TDI诱导的职业性哮喘诱导痰中往往以中性粒细胞为主。目前以TDI为致敏原诱导哮喘模型的研究很多。因此本实验第一个目的是建立一种TDI诱哮喘小鼠模型,为研究中性粒细胞哮喘模型的机制打下坚实的基础。 支气管哮喘是一种免疫调节异常性疾病,这一点已经取得了普遍共识,有关哮喘免疫调节紊乱的机制得到最广泛关注的是“卫生假说”,其核心内容是TH1/TH2细胞因子平衡学说,TH1/TH2细胞因子有相互制约彼此表型分化的功能和特性。当TH1炎症占优势是就可以抑制TH2炎症,所以在西方国家卫生条件较好的情况下,当婴幼儿时期接触细菌病毒等等有害物质过少,就会导致TH2细胞的过度分化,从而导致哮喘疾病的发生。后来的研究发现同样的生活方式的人群以TH2炎症为主的哮喘高发,以TH1炎症为主的I型糖尿病发病率也很高。另一方面早前肠道寄生虫感染可以强有力的增加TH2炎症,但是它同样减少了哮喘的高发。也就是说简单的TH1/TH2炎症不能够完全解释哮喘的免疫发生机制。进一步的研究发现有一种机制也参与了哮喘的发生,发展,那就是免疫耐受,免疫耐受主要与CD4+调节性T细胞(Treg)有关,Treg、能够抑制THl炎症也能够抑制TH2炎症,哮喘的发生很可能去Treg功能的缺陷也是有关系的。一系列临床研究发现孕期口服维生素D能够有效的降低孩子出生后哮喘发病率,并且有研究提示,口服维生素D能够有效的降低吸入激素(ICS)的使用量。动物与细胞水平的研究提示维生素D可能通过以下途径影响哮喘的发生与发展。首先维生素D增强气道固有免疫,可以促进一些抗菌肽的分泌。其次是维生素D对哮喘的效应细胞有影响,例如可以抑制TH2细胞的成熟分化,抑制IL-4, IL-5等炎症因子的释放。最后维生素D可以有效的促进Treg细胞的活化,Treg细胞能够有效的调节TH2细胞与TH1细胞之间的平衡,特别是抑制TH2细胞的作用。 近年来支气管上皮在哮喘的作用逐渐受到重视,支气管上皮是机体与外界接触的直接屏障,研究发现致敏原等有害物质可以穿过支气管上皮屏障被支气管粘膜下抗原提细胞捕获,并提呈给T淋巴细胞从而诱发哮喘炎症,支气管上皮细胞屏障主要包括支气管上皮细胞,及上皮细胞之间的连接装置,而这些连接装置主要包括紧密连接,粘附连接及桥粒连接。紧密连接是对支气管上皮物理屏障的直接结构基础。进一步的研究发现,粘附连接中的E-cadherin在上皮的屏障中起到重要的作用,首先是它作为上皮屏障的一种连接蛋白,更重要的是它可以调节紧密连接蛋白,如ZO-l,ocludin,clauding-2等紧密连接蛋白的生成与分布。采用RNA干扰技术发现,下调E-cadherin的表达水平能偶激活NF-kabar B通路,而NF-kabar B通路可以促进哮喘中很多炎症因子的分泌。研究显示E-cadherin可以直接或者间接的抑制TH2细胞的分化及成熟及其作用,同时促进Treg的分化成熟,来自于纤维支气管镜活检获得的临床标本发现,E-cadherin在哮喘病人支气管上皮表达降低,且其降低的程度与疾病的严重程度正相关。 目前的研究发现维生素D对上皮的屏障功能也是有影响的,如维生素D可以通过促进肠上皮E-cadherin, ZO-1等等连接蛋白的生成与分布降低上皮的通透性。同样的研究结果在对角膜上皮细胞通透性研究的过程中也得到证实。那么维生素D是否对哮喘小鼠支气管上皮E-cadherin有影响,如果有影响,我们推测维生素D对支气管上皮E-cadherin的影响可能是它影响哮喘炎症的一个机制。所以本实验的第二个目的是观察哮喘炎症及E-cadherin在TDI诱导的哮喘小鼠模型中的改变,并且观察、Vitamin D的干预作用。 研究内容和方法： 第一部分：参考国外研究者的建立模型方法建立TDI诱导的哮喘小鼠模型 第二部分：在此模型的基础之上给予维生素D干预(腹腔注射),观察哮喘炎症及上皮E-cadherin的改变。 结果： 参考国外学者建立哮喘模型的方法,第1天,8天给小鼠耳背部皮肤致敏,第15天口咽部吸入激发。我们成功建立了哮喘模型,其主要特征是,肺泡灌洗液炎症细胞总数(t=-7.956,P0.001)、中性粒细胞(t=-9.460,P0.001)较对照组显著增加,无嗜酸性粒细胞浸润,血清IgE浓度(t=-5.510,P0.001),淋巴培养上清IL-4(t=--22.989,P0.001)及IFN-γ(t=-15.028,P0.001)浓度显著升高,气道反应性增高。但是作为哮喘模型的一个很重要的特点支气管及血管周围炎症细胞浸润在我们的模型中并未发现。因此我们通过增加激发次数来改进这一模型。我们又分别观察第15天(1次激发组),15,18天(2次激发组),及15,18,21天(3次激发组)小鼠模型的情况发现随着激发次数的增加,哮喘小鼠肺泡灌洗液炎症细胞总数(F=37.092,P0.001)、中性粒细胞数(F=19.473,P0.001)、嗜酸性粒细胞数显著增加(F=78.890,P0.001),同时第3次激发后发现明显的支气管及血管周围炎症细胞浸润同时伴有支气管上皮的增生。接下来为了进一步完善模型,我们又对这一模型的炎症持续时间做了观察,观察时间点为第3次激发后的24h,72h及120h,发现随着观察时间点的而延长,肺泡灌洗液炎症细胞总数(F=26.929,P0.001)、中性粒细胞(F=5.892,P=0.010)、嗜酸性粒细胞(F=8.921,P0.010)显著下降,但是120h这一时间点的各项炎症指标仍然高于对照组。 腹腔注射维生素D后发现,IL-4(F=94.727,P0.001), IFN-γ (F=42.996,P0.001)及血清IgE (F=120.414,P0.001),肺泡灌洗液炎症细胞总数(F=56.729,P0.001),嗜酸性粒细胞总数(F=4.972,P0.001)等哮喘组较对照组显著升高,维生素D较哮喘组显著下降,中性粒细胞哮喘组较对照组显著升高,但维生素D组较哮喘组无明显变化。维生素D组肺组织支气管及血管周围炎症及气道高反应性也较哮喘组减轻。免疫组化显示：E-cadherin主要分布在支气管上皮细胞与上皮细胞的连接处及基底部,TDI诱导的中性粒细胞哮喘小鼠上皮细胞与上皮细胞连接处及基底部表达明显减少,维生素D组小鼠较哮喘组小鼠有所恢复。 结论： 1,我们成功的建立并优化了TDI诱导中性粒细胞哮喘模型 2,给予维生素D干预对哮喘炎症具有抑制作用,但是不能够减少肺泡灌洗液中中性粒细胞数量 3,维生素D有可能通过改善哮喘小鼠支气管上皮E-cadherin的分布抑制TDI诱导的哮喘炎症。
[Abstract]:At present, hormones are the most effective drugs for the treatment of asthma, but there are still some asthmatics in spite of the use of large doses of hormones that can not be effectively controlled. This part of the asthma is called steroid resistant asthma. Studies have shown that these patients are induced by sputum and are mainly neutrophils. Because the asthma phenotype is based on the classification of patients, asthma patients can be more individualized. There are four main types of asthma phenotypes: eosinophil, neutrophils, granulocytic and granulocytic type. Granulocyte asthma is not clear about the cause of inhaled hormone resistance. Neutrophils are a cause of hormone resistance, or it is only a manifestation of the hormone resistance to asthma, and there is still a lot of controversy. Preliminary research shows that hormone inhibition of neutrophils may be a cause of hormone resistance. The main reason for the study of neutrophil asthma phenotype is that there is no reliable animal model. The most commonly used sensitization of asthma animal models to date is chicken egg white protein (OVA), and OVA induced asthma model is mainly eosinophil, and OVA induced asthma for 100 years, which makes the researchers on the hair of asthma. The understanding of the mechanism of the disease has made great progress and the development of the drug has been successful to a certain extent. Therefore, we are trying to establish a stable and reliable model of asthma animal as a platform to study neutrophil type asthma.
Toluene diisocyanate (TDI), a common industrial raw material, is the most important cause of occupational asthma, but as our lifestyle changes, this industrial raw material, mainly used in paint and foam materials, has become closely related to all of us after birth. Clinical studies have found TDI induced occupational asthma. The induced sputum is often dominated by neutrophils. There are many studies on the model of asthma induced by TDI as allergens. Therefore, the first aim of this experiment is to establish a model of TDI induced asthma in mice, and to lay a solid foundation for the study of the mechanism of neutrophil asthma model.
Bronchial asthma is a kind of immunoregulatory abnormal disease, which has gained universal consensus. The most widely concerned mechanism of asthma immune regulation disorder is "health hypothesis", its core is TH1/TH2 cytokine balance theory, TH1/TH2 cytokines have the function and characteristics that restrict the differentiation of each other with each other. When T H1 inflammation is the dominant factor in the inhibition of TH2 inflammation, so in the western countries with good health conditions, when young children are exposed to bacterial virus and other harmful substances, the excessive differentiation of TH2 cells will lead to the occurrence of asthma. The incidence of asthma is high, and the incidence of type I diabetes mainly with TH1 inflammation is also high. On the other hand, early intestinal parasitic infection can increase TH2 inflammation strongly, but it also reduces the high incidence of asthma. That is to say, simple TH1/TH2 inflammation can not fully explain the pathogenesis of asthma. The mechanism is also involved in the development of asthma, which is immune tolerance. Immune tolerance is mainly associated with CD4+ regulatory T cells (Treg), Treg, which inhibits THl inflammation and inhibits TH2 inflammation. The occurrence of asthma is likely to be associated with the deficiency of Treg function. A series of clinical studies have found that oral vitamin D in pregnancy can be effective. Reduce the incidence of asthma in children after birth, and studies have suggested that oral vitamin D can effectively reduce the use of inhaled hormone (ICS). Animal and cell level studies suggest that vitamin D may affect the development and development of asthma through the following pathways. First, vitamin D increases the innate immunity of the airway, which can promote some antimicrobial peptides. Secretion. Secondly, vitamin D has an effect on the effector cells of asthma. For example, it can inhibit the maturation of TH2 cells and inhibit the release of IL-4, IL-5 and other inflammatory factors. Finally, vitamin D can effectively promote the activation of Treg cells, and Treg cells can effectively regulate the balance between TH2 cells and TH1 cells, especially the inhibition of TH2 cells. Use.
In recent years, the role of bronchial epithelium in asthma is becoming more and more important. Bronchial epithelium is a direct barrier to the body's contact with the outside world. It is found that the harmful substances such as the sensitized source can be caught by the bronchial epithelial barrier by the submucosal antigen cells of the bronchial mucosa and presented to the T lymphocytic cells to induce asthma inflammation and bronchial epithelial cells. The barrier consists mainly of the connection devices between the bronchial epithelial cells and the epithelial cells, which mainly include close connections, adhesion connections and bridging connections. Close connection is the direct structural basis for the physical barrier of the bronchial epithelium. Further studies have found that the E-cadherin in the attached junction plays a role in the epithelial barrier. The important role is that it is a connexin in the epithelial barrier, and more importantly, it can regulate the formation and distribution of tight connexin, such as ZO-l, ocludin, clauding-2 and so on. Using RNA interference technique, the downregulation of E-cadherin expression level can activate the NF-kabar B pathway, and NF-kabar B pathway can be promoted. A number of inflammatory factors are secreted in asthma. Studies have shown that E-cadherin can directly or indirectly inhibit the differentiation and maturation of TH2 cells and promote the differentiation and maturation of Treg. Clinical specimens obtained from fiberoptic bronchoscopic biopsy have found that the expression of E-cadherin in bronchial epithelium in asthmatic patients is reduced, and the decrease in the process is reduced. The degree of the disease is positively related to the severity of the disease.
Current studies have found that vitamin D is also affected by the barrier function of the epithelium, such as vitamin D can reduce the permeability of epithelium by promoting the formation and distribution of E-cadherin, ZO-1 and other connexin in the intestinal epithelium. The same results are also confirmed in the study of corneal epithelial cell permeability. Then vitamin D is the same No effect on bronchial epithelial E-cadherin in asthmatic mice. If there is an impact, we speculate that the effect of vitamin D on bronchial epithelial E-cadherin may be a mechanism that affects asthma inflammation. So the second aim of this study was to observe the changes in asthma and E-cadherin in the model of asthma induced asthma in mice and to observe the changes in the model of asthma induced by TDI. The intervention of Vitamin D.
Research contents and methods:
The first part is to establish TDI induced asthma mouse model with reference to foreign researchers.
The second part: Based on this model, vitamin D intervention (intraperitoneal injection) was used to observe the changes of asthma inflammation and epithelial E-cadherin.
Result锛，

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