白细胞介素-17A及骨桥蛋白在肺结核治疗过程中免疫调节机制研究

发布时间：2017-12-31 04:14

本文关键词：白细胞介素-17A及骨桥蛋白在肺结核治疗过程中免疫调节机制研究　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景与实验目的:结核病(Tuberculosis,TB)是由结核分枝杆菌(Mycobacterium,tuberculosis,M.TB)感染引起的传染性疾病,TB的发生和发展与患者的免疫功能,尤其是细胞免疫功能密切相关,其中主要效应细胞为T淋巴细胞。CD4幼稚细胞可分化为Th1、Th2和Th17亚群,IL-17为Th17细胞分泌的主要的细胞因子。有研究发现,在结核性胸水,肺结核支气管灌洗液和外周血中可检测到IL-17 mRNA的表达,提示Th17细胞及IL-17参与了结核的病理过程。而在许多功能上,Th17细胞同调节性T淋巴细胞(Regulatory T cell,Treg)密切相关,控制着体内自身免疫反应,在免疫耐受和动态平衡中起中心作用。Treg细胞数量和功能出现受损,免疫耐受就被打破。在许多慢性炎症,包括结核感染引起的慢性炎症中,IL-17和IFN-γ相互作用可以引起病理性损伤。骨桥蛋白(Osteopontin,OPN)属于细胞外基质蛋白,是可溶性的磷酸化糖蛋白。有研究发现,OPN可以作为细胞因子的一种参与类风湿性关节炎、肝炎、肝癌等多种疾病的发生发展。另外,OPN可通过增加IL-12和IFN-γ分泌而加重结核杆菌感染后肺部的病理性损伤。体外培养巨噬细胞株,感染结核杆菌之后,OPN在培养上清中的水平大幅度增加。这些表明OPN可能参与了 TB的发生发展过程。Th17细胞、Treg细胞、Th17和Treg细胞相关的细胞因子以及OPN对于结核感染的发生、发展及预后都有重要意义,但目前关于疗效和预后的动态观察报道较少,而且Th17细胞、IL-17、Treg细胞在肺结核发病过程中的作用尚存在争议。本课题将通过观察和分析初治痰涂片抗酸染色(Acid Fast Bacilli-smear,AFB)阳性的肺结核患者和经抗结核治疗AFB转阴后外周血IL-17、IL-23、IL-6、IFN-γ、IL-10、OPN、IP-10的水平,以及Treg细胞在治疗过程中的动态变化,进一步阐明肺结核治疗过程中细胞免疫的特征的变化,并探索其中的机制,同时找出评价肺结核治疗效果的可靠指标。研究方法:收集、整理和分析20例AFB阳性的肺结核患者的临床资料和样本,以及对应的20例健康者的资料和样本。流式细胞技术检测20例AFB阳性肺结核患者外周血单个核细胞(PBMCs)中T淋巴细胞、Treg细胞的比例,以及产生IL-17A细胞的类型和比例,酶联免疫斑点实验(Enzyme-linked Immunospot Assay,ELISPOT)检测产生IL-17A的细胞数量,酶联免疫吸附实验(Enzyme-linked immunosorbent assay,ELISA)检测20例AFB阳性肺结核患者治疗前后体内细胞因子IL-17A、IL-23、IL-6、IFN-γ、IL-10,以及OPN、IP-10的水平。同样的方法检测对应的20例健康者相应指标的水平。体外培养肺泡上皮细胞株(A549),加入BCG、IL-17A、IL-10、OPN等刺激,观察细胞株的变化,ELISA法检测培养上清中细胞和因子乳酸脱氢酶(Lactate Dehydrogenase,LDH)的水平,研究IL-17A、IL-10、OPN等在结核的发生发展过程中的不同作用,并探索其中可能的机制。体外培养健康者和肺结核患者PBMC,在BCG和OPN等刺激后24h,ELISA方法检测培养上清中细胞因子的水平。研究结果:第一部分AFB阳性患者表达IL-17A的能力较健康对照组明显增加,而经抗结核治疗,痰涂片转阴后,患者IL-17A的水平虽明显下降,但是仍然高于健康对照的水平。同时与健康对照组比较,AFB阳性患者表达IL-23、IL-6、IFN-y的能力亦显著增加。流式细胞技术分析,在AFB阳性的患者中产生IL-17A细胞类型除了经典T细胞(CD3+CD56-)外,CD3+CD56+NKTlike细胞是另一个主要来源。Treg细胞以及其分泌的细胞因子IL-10能够抑制Th17细胞分泌IL-17A。IL-17A与血液中C反应蛋白(C-reactive protein,CRP)和红细胞沉降率(Eythrocyte Sedimentation Rate,ESR)呈正相关,而与IL-10呈负相关。体外实验发现IL-17A能够诱导肺泡上皮细胞株的凋亡增加,而且能够加重BCG引起的肺泡上皮细胞损伤,而IL-10能够抑制IL-17A对肺泡上皮细胞的损伤作用。第二部分OPN血浆中的水平在AFB阳性的患者中较健康对照组明显的升高,AFB转阴后又明显的下降,AFB阳性合并结核性胸水(TPE)患者OPN的表达水平明显高于AFB阳性未合并TPE的患者的水平。并且OPN与CRP呈明显正相关。OPN能够促进γ干扰素诱导蛋白10(IFN-γ-induced protein 10,IP-10)的表达,IP-10在痰涂片阳性的患者中表达也明显的升高。OPN和IP-10只有在痰涂片转阴后才有明显的下降。同时,BCG能够促进肺泡上皮细胞分泌OPN,而OPN能够促进肺泡上皮细胞分泌IP-10,同样的OPN能够促进健康者和肺结核患者PBMC产生IP-10、IL-12、IFN-y,而加入IFN-y中和抗体后,PBMC分泌IP-10和IL-12的能力显著降低。结论:在结核分枝杆菌感染过程中,IL-17A能够诱导肺损伤,CD3+CD56+ NKT like细胞为AFB阳性的肺结核患者IL-17A的重要来源。在结核的发展过程中,Treg细胞及其分泌的细胞因子IL-10可以抑制IL-17A引起的肺损伤。OPN能够诱导结核感染引起的肺损伤,OPN和IP-10可能成为判断结核治疗效果的新指标。
[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.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R521

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