本文關(guān)鍵詞：白細(xì)胞介素-17A及骨橋蛋白在肺結(jié)核治療過(guò)程中免疫調(diào)節(jié)機(jī)制研究　出處：《浙江大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 痰涂片陽(yáng)性肺結(jié)核 調(diào)節(jié)性T細(xì)胞 自然殺傷T細(xì)胞 白介素-17 γ干擾素誘導(dǎo)蛋白-10 骨橋蛋白

【摘要】：研究背景與實(shí)驗(yàn)?zāi)康?結(jié)核病(Tuberculosis,TB)是由結(jié)核分枝桿菌(Mycobacterium,tuberculosis,M.TB)感染引起的傳染性疾病,TB的發(fā)生和發(fā)展與患者的免疫功能,尤其是細(xì)胞免疫功能密切相關(guān),其中主要效應(yīng)細(xì)胞為T(mén)淋巴細(xì)胞。CD4幼稚細(xì)胞可分化為T(mén)h1、Th2和Th17亞群,IL-17為T(mén)h17細(xì)胞分泌的主要的細(xì)胞因子。有研究發(fā)現(xiàn),在結(jié)核性胸水,肺結(jié)核支氣管灌洗液和外周血中可檢測(cè)到IL-17 mRNA的表達(dá),提示Th17細(xì)胞及IL-17參與了結(jié)核的病理過(guò)程。而在許多功能上,Th17細(xì)胞同調(diào)節(jié)性T淋巴細(xì)胞(Regulatory T cell,Treg)密切相關(guān),控制著體內(nèi)自身免疫反應(yīng),在免疫耐受和動(dòng)態(tài)平衡中起中心作用。Treg細(xì)胞數(shù)量和功能出現(xiàn)受損,免疫耐受就被打破。在許多慢性炎癥,包括結(jié)核感染引起的慢性炎癥中,IL-17和IFN-γ相互作用可以引起病理性損傷。骨橋蛋白(Osteopontin,OPN)屬于細(xì)胞外基質(zhì)蛋白,是可溶性的磷酸化糖蛋白。有研究發(fā)現(xiàn),OPN可以作為細(xì)胞因子的一種參與類風(fēng)濕性關(guān)節(jié)炎、肝炎、肝癌等多種疾病的發(fā)生發(fā)展。另外,OPN可通過(guò)增加IL-12和IFN-γ分泌而加重結(jié)核桿菌感染后肺部的病理性損傷。體外培養(yǎng)巨噬細(xì)胞株,感染結(jié)核桿菌之后,OPN在培養(yǎng)上清中的水平大幅度增加。這些表明OPN可能參與了 TB的發(fā)生發(fā)展過(guò)程。Th17細(xì)胞、Treg細(xì)胞、Th17和Treg細(xì)胞相關(guān)的細(xì)胞因子以及OPN對(duì)于結(jié)核感染的發(fā)生、發(fā)展及預(yù)后都有重要意義,但目前關(guān)于療效和預(yù)后的動(dòng)態(tài)觀察報(bào)道較少,而且Th17細(xì)胞、IL-17、Treg細(xì)胞在肺結(jié)核發(fā)病過(guò)程中的作用尚存在爭(zhēng)議。本課題將通過(guò)觀察和分析初治痰涂片抗酸染色(Acid Fast Bacilli-smear,AFB)陽(yáng)性的肺結(jié)核患者和經(jīng)抗結(jié)核治療AFB轉(zhuǎn)陰后外周血IL-17、IL-23、IL-6、IFN-γ、IL-10、OPN、IP-10的水平,以及Treg細(xì)胞在治療過(guò)程中的動(dòng)態(tài)變化,進(jìn)一步闡明肺結(jié)核治療過(guò)程中細(xì)胞免疫的特征的變化,并探索其中的機(jī)制,同時(shí)找出評(píng)價(jià)肺結(jié)核治療效果的可靠指標(biāo)。研究方法:收集、整理和分析20例AFB陽(yáng)性的肺結(jié)核患者的臨床資料和樣本,以及對(duì)應(yīng)的20例健康者的資料和樣本。流式細(xì)胞技術(shù)檢測(cè)20例AFB陽(yáng)性肺結(jié)核患者外周血單個(gè)核細(xì)胞(PBMCs)中T淋巴細(xì)胞、Treg細(xì)胞的比例,以及產(chǎn)生IL-17A細(xì)胞的類型和比例,酶聯(lián)免疫斑點(diǎn)實(shí)驗(yàn)(Enzyme-linked Immunospot Assay,ELISPOT)檢測(cè)產(chǎn)生IL-17A的細(xì)胞數(shù)量,酶聯(lián)免疫吸附實(shí)驗(yàn)(Enzyme-linked immunosorbent assay,ELISA)檢測(cè)20例AFB陽(yáng)性肺結(jié)核患者治療前后體內(nèi)細(xì)胞因子IL-17A、IL-23、IL-6、IFN-γ、IL-10,以及OPN、IP-10的水平。同樣的方法檢測(cè)對(duì)應(yīng)的20例健康者相應(yīng)指標(biāo)的水平。體外培養(yǎng)肺泡上皮細(xì)胞株(A549),加入BCG、IL-17A、IL-10、OPN等刺激,觀察細(xì)胞株的變化,ELISA法檢測(cè)培養(yǎng)上清中細(xì)胞和因子乳酸脫氫酶(Lactate Dehydrogenase,LDH)的水平,研究IL-17A、IL-10、OPN等在結(jié)核的發(fā)生發(fā)展過(guò)程中的不同作用,并探索其中可能的機(jī)制。體外培養(yǎng)健康者和肺結(jié)核患者PBMC,在BCG和OPN等刺激后24h,ELISA方法檢測(cè)培養(yǎng)上清中細(xì)胞因子的水平。研究結(jié)果:第一部分AFB陽(yáng)性患者表達(dá)IL-17A的能力較健康對(duì)照組明顯增加,而經(jīng)抗結(jié)核治療,痰涂片轉(zhuǎn)陰后,患者IL-17A的水平雖明顯下降,但是仍然高于健康對(duì)照的水平。同時(shí)與健康對(duì)照組比較,AFB陽(yáng)性患者表達(dá)IL-23、IL-6、IFN-y的能力亦顯著增加。流式細(xì)胞技術(shù)分析,在AFB陽(yáng)性的患者中產(chǎn)生IL-17A細(xì)胞類型除了經(jīng)典T細(xì)胞(CD3+CD56-)外,CD3+CD56+NKTlike細(xì)胞是另一個(gè)主要來(lái)源。Treg細(xì)胞以及其分泌的細(xì)胞因子IL-10能夠抑制Th17細(xì)胞分泌IL-17A。IL-17A與血液中C反應(yīng)蛋白(C-reactive protein,CRP)和紅細(xì)胞沉降率(Eythrocyte Sedimentation Rate,ESR)呈正相關(guān),而與IL-10呈負(fù)相關(guān)。體外實(shí)驗(yàn)發(fā)現(xiàn)IL-17A能夠誘導(dǎo)肺泡上皮細(xì)胞株的凋亡增加,而且能夠加重BCG引起的肺泡上皮細(xì)胞損傷,而IL-10能夠抑制IL-17A對(duì)肺泡上皮細(xì)胞的損傷作用。第二部分OPN血漿中的水平在AFB陽(yáng)性的患者中較健康對(duì)照組明顯的升高,AFB轉(zhuǎn)陰后又明顯的下降,AFB陽(yáng)性合并結(jié)核性胸水(TPE)患者OPN的表達(dá)水平明顯高于AFB陽(yáng)性未合并TPE的患者的水平。并且OPN與CRP呈明顯正相關(guān)。OPN能夠促進(jìn)γ干擾素誘導(dǎo)蛋白10(IFN-γ-induced protein 10,IP-10)的表達(dá),IP-10在痰涂片陽(yáng)性的患者中表達(dá)也明顯的升高。OPN和IP-10只有在痰涂片轉(zhuǎn)陰后才有明顯的下降。同時(shí),BCG能夠促進(jìn)肺泡上皮細(xì)胞分泌OPN,而OPN能夠促進(jìn)肺泡上皮細(xì)胞分泌IP-10,同樣的OPN能夠促進(jìn)健康者和肺結(jié)核患者PBMC產(chǎn)生IP-10、IL-12、IFN-y,而加入IFN-y中和抗體后,PBMC分泌IP-10和IL-12的能力顯著降低。結(jié)論:在結(jié)核分枝桿菌感染過(guò)程中,IL-17A能夠誘導(dǎo)肺損傷,CD3+CD56+ NKT like細(xì)胞為AFB陽(yáng)性的肺結(jié)核患者IL-17A的重要來(lái)源。在結(jié)核的發(fā)展過(guò)程中,Treg細(xì)胞及其分泌的細(xì)胞因子IL-10可以抑制IL-17A引起的肺損傷。OPN能夠誘導(dǎo)結(jié)核感染引起的肺損傷,OPN和IP-10可能成為判斷結(jié)核治療效果的新指標(biāo)。
[Abstract]:Objective to study the background and experimental tuberculosis (Tuberculosis, TB) by Mycobacterium tuberculosis (Mycobacterium, tuberculosis, M.TB) infection of infectious diseases caused by the immune function of patients with the occurrence and development of TB, especially the cellular immune function is closely related to the main effect of T cell.CD4 cells of immature cells can differentiate into Th1. The Th2 and Th17 subgroups, the main cytokine IL-17 secretion of Th17 cells. Studies have found that in tuberculous pleural effusion, detected the expression of IL-17 mRNA of pulmonary tuberculosis in bronchoalveolar lavage fluid and peripheral blood, in Th17 cells and IL-17 is involved in the pathological process of tuberculosis. But in many functions. Th17 cells with regulatory T cells (Regulatory T cell, Treg) is closely related to control the body's own immune response, the immune tolerance and homeostasis plays a central role in.Treg cell number and function damaged, free Immune tolerance is broken. In many chronic inflammation, including chronic inflammation caused by tuberculosis, IL-17 and IFN- gamma interaction can cause pathological damage. Osteopontin (Osteopontin, OPN) belongs to the extracellular matrix protein is phosphorylated glycoprotein soluble. Studies have found that OPN can be used as a kind of participation in rheumatoid arthritis inflammatory cytokines, hepatitis, occurrence and development of liver cancer and other diseases. In addition, OPN can aggravate the pathological damage of lung tuberculosis infection by adding IL-12 and IFN- secretion. In vitro macrophage, after infection of Mycobacterium tuberculosis, the OPN level in the culture supernatant increased significantly. These suggest that OPN may be involved in the occurrence and development of.Th17 cells, TB Treg cells, Th17 Treg cells and related cytokines and OPN for tuberculosis infection, development and prognosis has important significance, but. The dynamic observation about curative effect and prognosis of less, and Th17 cells, IL-17 Treg cells in the pathogenesis of pulmonary tuberculosis is still controversial. This paper through observation and analysis of initial treatment smear acid fast staining (Acid Fast, Bacilli-smear, AFB) - positive tuberculosis patients and treatment with anti TB AFB. Peripheral blood IL-17, IL-23, IL-6, IL-10, OPN, IFN- gamma, IP-10 level, and the dynamic changes of Treg cells in the treatment process, to clarify the change characteristics of immune cells in treatment of pulmonary tuberculosis, and to explore the mechanism, and find out the reliable index to evaluate therapeutic efficacy of pulmonary tuberculosis. Methods: to collect, collate and analyze the clinical data and samples of 20 cases of AFB positive patients with pulmonary tuberculosis, and the corresponding sample data and 20 healthy controls. Flow cytometry in 20 patients with AFB positive pulmonary tuberculosis in peripheral blood Mononuclear cells (PBMCs) in T cells, Treg cells and IL-17A cell ratio, type and proportion of ELISPOT (Enzyme-linked Immunospot, Assay, ELISPOT) to detect the quantity of cells producing IL-17A, enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent, assay, ELISA) before and after AFB was positive in 20 cases of pulmonary tuberculosis patients cytokine IL-17A, IL-23, IL-6, IFN-, IL-10, gamma, OPN, IP-10. 20 healthy subjects were also detected corresponding index level. In vitro cultured alveolar epithelial cell line (A549), IL-17A, IL-10, joined BCG, OPN stimulation, observe the change of cell line, ELISA we detected the cytokines and lactate dehydrogenase in the supernatant (Lactate Dehydrogenase, LDH) level of IL-17A, IL-10, OPN etc. have different role in the process of development in the occurrence of tuberculosis, and to explore its possible mechanism. PBMC patients and healthy subjects of pulmonary tuberculosis in vitro, in BCG and OPN after the stimulation of 24h, detection of cytokines in culture supernatants ELISA method. Results: the first part is the ability of IL-17A expression in AFB positive patients than in the healthy control group increased significantly, and after anti tuberculosis treatment, sputum smear negative patients, the level of IL-17A although significantly decreased, but still higher than that of the control level. At the same time compared with the healthy control group, IL-23, AFB expression in IL-6 positive patients, the ability of IFN-y also increased significantly. Analysis of flow cytometry in AFB positive patients have IL-17A cell types in addition to the classic T cell (CD3+CD56-), CD3+CD56+NKTlike cell is another a major source of.Treg cells and the secretion of cytokines of IL-10 could inhibit the secretion of Th17 cells and IL-17A.IL-17A in the blood of C reactive protein (C-reactive protein, CRP) and erythrocyte sedimentation rate (Eythrocyt E Sedimentation Rate, ESR) were positively correlated, and negatively correlated with IL-10. In vitro experiments showed that IL-17A could induce apoptosis of alveolar epithelial cells increased, the damage of alveolar epithelial cells and can increase BCG induced IL-10 damage, can inhibit the role of IL-17A in alveolar epithelial cells. The second part of the OPN plasma level in AFB positive patients compared with the healthy control group significantly increased, decreased markedly after AFB negative, AFB positive patients with tuberculous pleural effusion (TPE) expression level in OPN patients was significantly higher than that of non AFB positive patients with TPE levels. And OPN and CRP showed a positive correlation with.OPN can promote the interferon inducible protein 10 (IFN- gamma -induced protein 10, IP-10) expression, the expression of IP-10 in patients with sputum smear positive also significantly increased.OPN and IP-10 decreased significantly only in sputum smear negative. At the same time, BCG can promote lung Bubble epithelial cells secrete OPN and OPN could promote the IP-10 secretion of alveolar epithelial cells, the same OPN can promote the healthy persons and PBMC patients with pulmonary tuberculosis from IP-10, IL-12, IFN-y, and IFN-y after adding neutralizing antibody, PBMC secretion of IP-10 and IL-12 was decreased. Conclusion: Mycobacterium tuberculosis infection in IL-17A can induce lung injury, an important source of CD3+CD56+ NKT like cells into AFB positive patients with pulmonary tuberculosis IL-17A. In the process of development of tuberculosis, lung injury.OPN Treg cells and the secretion of IL-10 can inhibit IL-17A induced can induce lung injury caused by Mycobacterium tuberculosis infection, OPN and IP-10 may become a new index for judging the therapeutic effect tuberculosis.

【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】：2017
【分類號(hào)】：R521

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5 副主任醫(yī)師 韓詠霞;肺結(jié)核治療中病情反復(fù)怎么辦[N];衛(wèi)生與生活報(bào);2006年

6 副主任醫(yī)師 王長(zhǎng)柏;聯(lián)手挑戰(zhàn)人類健康[N];大眾衛(wèi)生報(bào);2001年

7 陳學(xué)梅 林恒;糖尿病合并肺結(jié)核治療中的幾個(gè)問(wèn)題[N];農(nóng)村醫(yī)藥報(bào)（漢）;2007年

8 楊春;結(jié)核久治不愈必有因[N];中國(guó)老年報(bào);2004年

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