本文選題：支氣管哮喘 + 中性粒細(xì)胞　； 參考：《南方醫(yī)科大學(xué)》2013年博士論文

【摘要】：目前激素是治療哮喘最有效的藥物,但是仍然有部分哮喘患者盡管使用了大劑量的激素,卻不能獲得有效的控制。這部分哮喘稱為激素抵抗型哮喘,有研究顯示這部分病人誘導(dǎo)痰,以中性粒細(xì)胞為主。目前對于哮喘表型的研究是熱點(diǎn)問題,因?yàn)橐韵硇蜑榛A(chǔ)對病人進(jìn)行分類,能夠使哮喘患者得到更加個性化的治療。以誘導(dǎo)痰炎癥細(xì)胞的種類對哮喘表型分類主要有一下四種類型：嗜酸性粒細(xì)胞型,中性粒細(xì)胞型,混合粒細(xì)胞型及少粒細(xì)胞型。目前對于為什么中性粒細(xì)胞哮喘對吸入激素抵抗原因還不清楚,中性粒細(xì)胞是激素抵抗的一個原因,還是說它只是激素抵抗哮喘患者的一個表現(xiàn),還存在很大爭議。初步的研究結(jié)果顯示,激素能夠抑制中性粒細(xì)胞凋亡可能是導(dǎo)致激素抵抗的一個原因。對于中性粒細(xì)胞哮喘表型的研究尚不深入,主要原因是沒有可靠的動物模型,迄今為止哮喘動物模型最常用的致敏原為雞卵清白蛋白(OVA),而OVA誘導(dǎo)的哮喘模型是以嗜酸性粒細(xì)胞為主,且應(yīng)用OVA誘導(dǎo)哮喘100多年來,使得研究者對于哮喘的發(fā)病機(jī)制的認(rèn)識有了很大的進(jìn)步,藥物的研發(fā)也獲得一定程度的成功。因此我們試圖建立一種穩(wěn)定可靠的哮喘動物模型作為平臺來研究中性粒細(xì)胞型哮喘。 甲苯二異氰酸酯(TDI),一種常用的工業(yè)原料,是成為職業(yè)性哮喘最為重要的誘因,但是隨著我們生活方式的改變,這種主要用于油漆及泡沫材料中的工業(yè)原料,已經(jīng)變得和我們每個人的生后都息息相關(guān)。臨床研究發(fā)現(xiàn)TDI誘導(dǎo)的職業(yè)性哮喘誘導(dǎo)痰中往往以中性粒細(xì)胞為主。目前以TDI為致敏原誘導(dǎo)哮喘模型的研究很多。因此本實(shí)驗(yàn)第一個目的是建立一種TDI誘哮喘小鼠模型,為研究中性粒細(xì)胞哮喘模型的機(jī)制打下堅實(shí)的基礎(chǔ)。 支氣管哮喘是一種免疫調(diào)節(jié)異常性疾病,這一點(diǎn)已經(jīng)取得了普遍共識,有關(guān)哮喘免疫調(diào)節(jié)紊亂的機(jī)制得到最廣泛關(guān)注的是“衛(wèi)生假說”,其核心內(nèi)容是TH1/TH2細(xì)胞因子平衡學(xué)說,TH1/TH2細(xì)胞因子有相互制約彼此表型分化的功能和特性。當(dāng)TH1炎癥占優(yōu)勢是就可以抑制TH2炎癥,所以在西方國家衛(wèi)生條件較好的情況下,當(dāng)嬰幼兒時期接觸細(xì)菌病毒等等有害物質(zhì)過少,就會導(dǎo)致TH2細(xì)胞的過度分化,從而導(dǎo)致哮喘疾病的發(fā)生。后來的研究發(fā)現(xiàn)同樣的生活方式的人群以TH2炎癥為主的哮喘高發(fā),以TH1炎癥為主的I型糖尿病發(fā)病率也很高。另一方面早前腸道寄生蟲感染可以強(qiáng)有力的增加TH2炎癥,但是它同樣減少了哮喘的高發(fā)。也就是說簡單的TH1/TH2炎癥不能夠完全解釋哮喘的免疫發(fā)生機(jī)制。進(jìn)一步的研究發(fā)現(xiàn)有一種機(jī)制也參與了哮喘的發(fā)生,發(fā)展,那就是免疫耐受,免疫耐受主要與CD4+調(diào)節(jié)性T細(xì)胞(Treg)有關(guān),Treg、能夠抑制THl炎癥也能夠抑制TH2炎癥,哮喘的發(fā)生很可能去Treg功能的缺陷也是有關(guān)系的。一系列臨床研究發(fā)現(xiàn)孕期口服維生素D能夠有效的降低孩子出生后哮喘發(fā)病率,并且有研究提示,口服維生素D能夠有效的降低吸入激素(ICS)的使用量。動物與細(xì)胞水平的研究提示維生素D可能通過以下途徑影響哮喘的發(fā)生與發(fā)展。首先維生素D增強(qiáng)氣道固有免疫,可以促進(jìn)一些抗菌肽的分泌。其次是維生素D對哮喘的效應(yīng)細(xì)胞有影響,例如可以抑制TH2細(xì)胞的成熟分化,抑制IL-4, IL-5等炎癥因子的釋放。最后維生素D可以有效的促進(jìn)Treg細(xì)胞的活化,Treg細(xì)胞能夠有效的調(diào)節(jié)TH2細(xì)胞與TH1細(xì)胞之間的平衡,特別是抑制TH2細(xì)胞的作用。 近年來支氣管上皮在哮喘的作用逐漸受到重視,支氣管上皮是機(jī)體與外界接觸的直接屏障,研究發(fā)現(xiàn)致敏原等有害物質(zhì)可以穿過支氣管上皮屏障被支氣管粘膜下抗原提細(xì)胞捕獲,并提呈給T淋巴細(xì)胞從而誘發(fā)哮喘炎癥,支氣管上皮細(xì)胞屏障主要包括支氣管上皮細(xì)胞,及上皮細(xì)胞之間的連接裝置,而這些連接裝置主要包括緊密連接,粘附連接及橋粒連接。緊密連接是對支氣管上皮物理屏障的直接結(jié)構(gòu)基礎(chǔ)。進(jìn)一步的研究發(fā)現(xiàn),粘附連接中的E-cadherin在上皮的屏障中起到重要的作用,首先是它作為上皮屏障的一種連接蛋白,更重要的是它可以調(diào)節(jié)緊密連接蛋白,如ZO-l,ocludin,clauding-2等緊密連接蛋白的生成與分布。采用RNA干擾技術(shù)發(fā)現(xiàn),下調(diào)E-cadherin的表達(dá)水平能偶激活NF-kabar B通路,而NF-kabar B通路可以促進(jìn)哮喘中很多炎癥因子的分泌。研究顯示E-cadherin可以直接或者間接的抑制TH2細(xì)胞的分化及成熟及其作用,同時促進(jìn)Treg的分化成熟,來自于纖維支氣管鏡活檢獲得的臨床標(biāo)本發(fā)現(xiàn),E-cadherin在哮喘病人支氣管上皮表達(dá)降低,且其降低的程度與疾病的嚴(yán)重程度正相關(guān)。 目前的研究發(fā)現(xiàn)維生素D對上皮的屏障功能也是有影響的,如維生素D可以通過促進(jìn)腸上皮E-cadherin, ZO-1等等連接蛋白的生成與分布降低上皮的通透性。同樣的研究結(jié)果在對角膜上皮細(xì)胞通透性研究的過程中也得到證實(shí)。那么維生素D是否對哮喘小鼠支氣管上皮E-cadherin有影響,如果有影響,我們推測維生素D對支氣管上皮E-cadherin的影響可能是它影響哮喘炎癥的一個機(jī)制。所以本實(shí)驗(yàn)的第二個目的是觀察哮喘炎癥及E-cadherin在TDI誘導(dǎo)的哮喘小鼠模型中的改變,并且觀察、Vitamin D的干預(yù)作用。 研究內(nèi)容和方法： 第一部分：參考國外研究者的建立模型方法建立TDI誘導(dǎo)的哮喘小鼠模型 第二部分：在此模型的基礎(chǔ)之上給予維生素D干預(yù)(腹腔注射),觀察哮喘炎癥及上皮E-cadherin的改變。 結(jié)果： 參考國外學(xué)者建立哮喘模型的方法,第1天,8天給小鼠耳背部皮膚致敏,第15天口咽部吸入激發(fā)。我們成功建立了哮喘模型,其主要特征是,肺泡灌洗液炎癥細(xì)胞總數(shù)(t=-7.956,P0.001)、中性粒細(xì)胞(t=-9.460,P0.001)較對照組顯著增加,無嗜酸性粒細(xì)胞浸潤,血清IgE濃度(t=-5.510,P0.001),淋巴培養(yǎng)上清IL-4(t=--22.989,P0.001)及IFN-γ(t=-15.028,P0.001)濃度顯著升高,氣道反應(yīng)性增高。但是作為哮喘模型的一個很重要的特點(diǎn)支氣管及血管周圍炎癥細(xì)胞浸潤在我們的模型中并未發(fā)現(xiàn)。因此我們通過增加激發(fā)次數(shù)來改進(jìn)這一模型。我們又分別觀察第15天(1次激發(fā)組),15,18天(2次激發(fā)組),及15,18,21天(3次激發(fā)組)小鼠模型的情況發(fā)現(xiàn)隨著激發(fā)次數(shù)的增加,哮喘小鼠肺泡灌洗液炎癥細(xì)胞總數(shù)(F=37.092,P0.001)、中性粒細(xì)胞數(shù)(F=19.473,P0.001)、嗜酸性粒細(xì)胞數(shù)顯著增加(F=78.890,P0.001),同時第3次激發(fā)后發(fā)現(xiàn)明顯的支氣管及血管周圍炎癥細(xì)胞浸潤同時伴有支氣管上皮的增生。接下來為了進(jìn)一步完善模型,我們又對這一模型的炎癥持續(xù)時間做了觀察,觀察時間點(diǎn)為第3次激發(fā)后的24h,72h及120h,發(fā)現(xiàn)隨著觀察時間點(diǎn)的而延長,肺泡灌洗液炎癥細(xì)胞總數(shù)(F=26.929,P0.001)、中性粒細(xì)胞(F=5.892,P=0.010)、嗜酸性粒細(xì)胞(F=8.921,P0.010)顯著下降,但是120h這一時間點(diǎn)的各項炎癥指標(biāo)仍然高于對照組。 腹腔注射維生素D后發(fā)現(xiàn),IL-4(F=94.727,P0.001), IFN-γ (F=42.996,P0.001)及血清IgE (F=120.414,P0.001),肺泡灌洗液炎癥細(xì)胞總數(shù)(F=56.729,P0.001),嗜酸性粒細(xì)胞總數(shù)(F=4.972,P0.001)等哮喘組較對照組顯著升高,維生素D較哮喘組顯著下降,中性粒細(xì)胞哮喘組較對照組顯著升高,但維生素D組較哮喘組無明顯變化。維生素D組肺組織支氣管及血管周圍炎癥及氣道高反應(yīng)性也較哮喘組減輕。免疫組化顯示：E-cadherin主要分布在支氣管上皮細(xì)胞與上皮細(xì)胞的連接處及基底部,TDI誘導(dǎo)的中性粒細(xì)胞哮喘小鼠上皮細(xì)胞與上皮細(xì)胞連接處及基底部表達(dá)明顯減少,維生素D組小鼠較哮喘組小鼠有所恢復(fù)。 結(jié)論： 1,我們成功的建立并優(yōu)化了TDI誘導(dǎo)中性粒細(xì)胞哮喘模型 2,給予維生素D干預(yù)對哮喘炎癥具有抑制作用,但是不能夠減少肺泡灌洗液中中性粒細(xì)胞數(shù)量 3,維生素D有可能通過改善哮喘小鼠支氣管上皮E-cadherin的分布抑制TDI誘導(dǎo)的哮喘炎癥。
[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錛，

本文編號：1906011