【摘要】：結(jié)核病是由結(jié)核分枝桿菌（Mycobacterium tuberculosis,Mtb）感染引起的以呼吸系統(tǒng)感染為主的慢性感染性疾病。由于結(jié)核病導(dǎo)致的組織損傷主要由機體的免疫系統(tǒng)介導(dǎo)，而且在結(jié)核病患者體內(nèi)往往存在Th1型免疫應(yīng)答受抑， Th2型免疫應(yīng)答水平異常升高的現(xiàn)象，結(jié)核病也被認為是一種免疫紊亂性疾病，機體免疫應(yīng)答的方向和水平在很大程度上決定著結(jié)核病的發(fā)展與轉(zhuǎn)歸。 中性粒細胞作為機體重要的固有免疫效應(yīng)細胞，是機體抵抗病原微生物感染的第一道防線。早期的研究認為中性粒細胞僅作為吞噬細胞在Mtb感染的早期參與抗結(jié)核病的固有免疫應(yīng)答，但近年來的研究顯示中性粒細胞還參與了結(jié)核病適應(yīng)性免疫的誘導(dǎo)和調(diào)節(jié)，對結(jié)核病免疫的發(fā)展有重要影響。 共刺激分子是一類表達于免疫細胞及免疫相關(guān)細胞上的蛋白，其介導(dǎo)的信號即為共刺激信號。共刺激信號對機體免疫應(yīng)答的調(diào)節(jié)是雙向的，通過正、負共刺激信號的調(diào)節(jié)使機體維持免疫平衡狀態(tài)。正性共刺激分子CD28、CD80、CD86等能激活T細胞，使其增殖、分化為效應(yīng)細胞，增強機體免疫應(yīng)答反應(yīng)，抵抗外來入侵的病原微生物；負性共刺激分子程序性死亡因子1（Programmeddeath1，PD-1）、程序性死亡因子1配體（Ligand of Programmed death1，PD-L1）則抑制T細胞增殖和產(chǎn)生細胞因子,抑制免疫應(yīng)答反應(yīng),防止過度的免疫損傷和自身免疫性疾病的發(fā)生,有利于維持機體的免疫自穩(wěn)。近年來有研究顯示中性粒細胞表面也會有共刺激分子的表達，提示我們中性粒細胞表達共刺激分子可能對機體抗結(jié)核病免疫有一定影響。 本研究通過對比分析活動性肺結(jié)核患者和健康人外周血中性粒細胞表面共刺激分子的表達水平及功能的差異，探討結(jié)核病患者外周血中性粒細胞共刺激分子的表達特性及細胞功能的改變。并通過Mtb體外感染的方法驗證了Mtb對中性粒細胞功能和共刺激分子表達的影響。在此基礎(chǔ)上，本研究采用特異性單抗阻斷PD-1/PD-L1信號途徑的方法，初步探討了該負性共刺激信號對中性粒細胞功能的調(diào)節(jié)作用。 第一部分結(jié)核病患者外周血中性粒細胞的功能及共刺激分子的表達特性 目的： 通過分析結(jié)核病患者中性粒細胞共刺激分子的表達水平及細胞功能的變化，探討結(jié)核病患者免疫功能異常與共刺激分子的關(guān)系。 方法： 收集30例活動性肺結(jié)核患者和20例健康體檢者的外周血，應(yīng)用流式細胞術(shù)檢測中性粒細胞表面共刺激分子的表達水平，CD62L的表達水平以及呼吸爆發(fā)產(chǎn)生ROS的水平；用硝酸還原酶法檢測中性粒細胞產(chǎn)生的NO；用Transwell裝置檢測中性粒細胞對IL-8的趨化能力。 結(jié)果： 與健康對照組相比，結(jié)核病患者外周血中性粒細胞具有以下特征：（1）CD86表達顯著降低（P 0.05），PD-L1分子表達顯著增加（P 0.01），而CD28、CD80、HLA-DR和PD-1均無顯著性變化（P0.05）；（2）經(jīng)PMA刺激后，呼吸爆發(fā)產(chǎn)生的ROS和NO水平顯著提高（P0.05）（；3）CD62L表達無顯著性差異（P0.05）；（4）對IL-8的趨化能力顯著降低（P 0.05）。 結(jié)論： 結(jié)核病患者中性粒細胞正性共刺激分子CD86表達下降，負性共刺激分子PD-L1表達升高，產(chǎn)生ROS和NO的量明顯增高，趨化能力有所降低。由此提示，共刺激分子表達失調(diào)可能會影響中性粒細胞功能。 第二部分結(jié)核分枝桿菌感染對中性粒細胞的功能及共刺激分子表達的影響 目的： 分析Mtb感染后中性粒細胞共刺激分子的表達水平及細胞功能的變化，探討Mtb感染對中性粒細胞的功能及共刺激分子表達的影響 方法： 收集20例健康體檢者的外周血，應(yīng)用流式細胞術(shù)檢測感染Mtb后中性粒細胞表面共刺激分子的表達水平，呼吸爆發(fā)產(chǎn)生ROS和CD62L的表達水平以及中性粒細胞的凋亡率；用硝酸還原酶法檢測感染Mtb后中性粒細胞產(chǎn)生的NO；使用Transwell裝置檢測感染Mtb后中性粒細胞對IL-8的趨化能力；用菌落計數(shù)（CFU）的方法觀察中性粒細胞對Mtb的殺菌能力。 結(jié)果： （1）與對照組相比，加入Mtb感染后中性粒細胞具有以下特征：①CD28、CD80、CD86、PD-1和PD-L1表達水平顯著提高（P 0.01），兩組中HLA-DR表達無顯著性差異（P0.05）；②產(chǎn)生ROS和NO的水平顯著提高（P 0.01）；③CD62L表達水平顯著降低（P 0.01）；④對IL-8的趨化能力顯著提高（P0.05）。 （2）MS和Mtb感染均可顯著上調(diào)中性粒細胞的凋亡水平，但與MS組相比，Mtb感染的中性粒細胞凋亡率更低（P 0.01）。 （3）中性粒細胞能夠有效地殺傷無致病性的MS，，卻不能殺傷有毒性的Mtb,導(dǎo)致Mtb在細胞內(nèi)繁殖。 結(jié)論： 1、Mtb感染能有效活化中性粒細胞，同時伴隨一系列共刺激分子的表達改變，但這些共刺激分子的變化是否與中性粒細胞的功能改變相關(guān)需要進一步的研究。 2、Mtb體外感染導(dǎo)致的中性粒細胞共刺激分子的改變與結(jié)核病患者外周血中性粒細胞表面共刺激分子的表達模式不完全一致，具體原因有待對結(jié)核病患者感染局部的中性粒細胞功能進行檢測分析后確定。 第三部分體外阻斷PD-1/PD-L1通路對中性粒細胞功能的影響 目的： 通過在體外對PD-1/PD-L1信號通路進行干預(yù)，探討該負性共刺激信號對中性粒細胞功能的調(diào)節(jié)作用。 方法： 收集15例健康體檢者的外周血，加入抗PD-L1單克隆抗體阻斷PD-1/PD-L1通路后，再加入Mtb感染30min，應(yīng)用流式細胞術(shù)檢測中性粒細胞ROS水平和CD62L的表達以及通過transwell趨化裝置檢測中性粒細胞趨化能力的改變。 結(jié)果： 與Mtb感染組相比，體外阻斷PD-1/PD-L1信號通路后,中性粒細胞產(chǎn)生ROS水平顯著增加（P 0.05），CD62L的表達水平顯著降低（P 0.05），中性粒細胞對IL-8的趨化能力提高（P 0.05）。 結(jié)論： 體外阻斷PD-1/PD-L1信號通路可以增強中性粒細胞的呼吸爆發(fā)功能，加速活化并增強其趨化能力，說明該負性共刺激信號會抑制中性粒細胞抗結(jié)核免疫的能力，這可能為臨床治療結(jié)核病開辟一條新途徑。
[Abstract]:Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb). Tissue damage caused by tuberculosis is mainly mediated by the body's immune system. Th1 immune response is often suppressed in patients with tuberculosis. Th2 immune response level Tuberculosis is also considered to be an immune disorder, and the direction and level of immune response to a large extent determines the development and outcome of tuberculosis.
Neutrophils, as important innate immune effector cells, are the first line of defense against pathogenic microbial infections. Early studies suggested that neutrophils, as phagocytes, participate only in the innate immune response to tuberculosis in the early stage of MTB infection, but recent studies have shown that neutrophils also participate in tuberculosis. Induction and regulation of adaptive immunity play an important role in the development of tuberculosis immunity.
Co-stimulatory molecule is a kind of protein expressed in immune cells and immune-related cells, and its mediated signal is co-stimulatory signal. Co-stimulatory signal regulates the immune response of the body in two directions. The body maintains immune balance through the regulation of positive and negative co-stimulatory signals. Cells, which proliferate and differentiate into effector cells, enhance the body's immune response and resist invasive pathogenic microorganisms; negative costimulatory molecule programmed death factor 1 (PD-1), programmed death factor 1 ligand (PD-L1) inhibits T cell proliferation and production of cytokines, inhibits immune response. Recent studies have shown that the expression of costimulatory molecules on the surface of neutrophils also suggests that the expression of costimulatory molecules in neutrophils may have some effect on the anti-tuberculosis immunity of the body.
In this study, the expression and function of costimulatory molecule on peripheral blood neutrophils were compared between active pulmonary tuberculosis patients and healthy people, and the expression characteristics and cell function of costimulatory molecule on peripheral blood neutrophils in patients with tuberculosis were investigated. On this basis, the regulation of the negative costimulatory signal on neutrophil function was preliminarily investigated by blocking the PD-1/PD-L1 signaling pathway with specific monoclonal antibodies.
Part one: function of peripheral blood neutrophils and expression characteristics of costimulatory molecules in TB patients
Objective:
To investigate the relationship between abnormal immune function and costimulatory molecule in tuberculosis patients by analyzing the expression level and cell function of neutrophil costimulatory molecule.
Method:
Peripheral blood samples from 30 patients with active pulmonary tuberculosis and 20 healthy subjects were collected. The expression of costimulatory molecules on the surface of neutrophils, the expression of CD62L and ROS were detected by flow cytometry, the production of NO by neutrophils was detected by nitrate reductase method, and neutrophils were detected by Transwell apparatus. The chemotaxis ability of cells to IL-8.
Result:
Compared with the healthy control group, the peripheral blood neutrophils of tuberculosis patients had the following characteristics: (1) CD86 expression was significantly decreased (P 0.05), PD-L1 molecule expression was significantly increased (P 0.01), while CD28, CD80, HLA-DR and PD-1 were not significantly changed (P 0.05); (2) ROS and NO levels in respiratory burst were significantly increased (P 0.05) after PMA stimulation (P 0.05); There was no significant difference in expression (P0.05); (4) the chemotactic ability of IL-8 was significantly decreased (P 0.05).
Conclusion:
The positive co-stimulatory molecule CD86 expression decreased, the negative co-stimulatory molecule PD-L1 expression increased, the production of ROS and NO increased, and the chemotaxis decreased in patients with tuberculosis.
The second part is the effect of Mycobacterium tuberculosis infection on the function of neutrophils and the expression of costimulatory molecules.
Objective:
To analyze the expression of costimulatory molecules in neutrophils and the changes of cell function after Mtb infection, and to explore the effect of Mtb infection on the function of neutrophils and the expression of costimulatory molecules.
Method:
The expression of costimulatory molecules on the surface of neutrophils, the expression of ROS and CD62L in respiratory burst and the apoptosis rate of neutrophils were detected by flow cytometry in peripheral blood of 20 healthy subjects. The chemotactic ability of neutrophils to IL-8 was tested after Mtb infection, and the bactericidal ability of neutrophils to Mtb was observed by colony count (CFU).
Result:
(1) Compared with the control group, the expression of CD28, CD80, CD86, PD-1 and PD-L1 increased significantly (P 0.01), but there was no significant difference in HLA-DR expression between the two groups (P 0.05); the expression of ROS and NO increased significantly (P 0.01); the expression of CD62L decreased significantly (P 0.01); and the chemotaxis of IL-8 decreased significantly (P 0.01). Ability was significantly improved (P0.05).
(2) Both MS and MTb infection significantly increased the level of apoptosis of neutrophils, but the apoptosis rate of neutrophils infected with Mtb was lower than that of MS group (P 0.01).
(3) Neutrophils can kill non-pathogenic MS effectively, but can not kill toxic Mtb, leading to the proliferation of Mtb in cells.
Conclusion:
1. Mtb infection can effectively activate neutrophils, accompanied by a series of co-stimulatory molecule expression changes, but whether these co-stimulatory molecule changes are related to the functional changes of neutrophils needs further study.
2. The changes of neutrophil costimulatory molecule induced by Mtb infection in vitro are inconsistent with the expression pattern of neutrophil surface costimulatory molecule in peripheral blood of tuberculosis patients. The specific reasons need to be determined after the detection and analysis of the local neutrophil function of tuberculosis patients.
The third part is the effect of blocking PD-1/PD-L1 pathway on neutrophil function in vitro.
Objective:
By interfering with the PD-1/PD-L1 signaling pathway in vitro, the regulatory effect of the negative costimulatory signal on neutrophil function was investigated.
Method:
After blocking PD-1/PD-L1 pathway with anti-PD-L1 monoclonal antibody and adding Mtb for 30 minutes, the expression of CD62L and ROS of neutrophils were detected by flow cytometry, and the chemotactic ability of neutrophils was detected by Transwell chemotactic apparatus.
Result:
Compared with Mtb infection group, the level of ROS production by neutrophils increased significantly (P 0.05), the expression of CD62L decreased significantly (P 0.05), and the chemotactic ability of neutrophils to IL-8 increased (P 0.05) after blocking PD-1/PD-L1 signaling pathway in vitro.
Conclusion:
Blocking the PD-1/PD-L1 signaling pathway in vitro can enhance the respiratory burst function of neutrophils, accelerate activation and enhance their chemotaxis. This indicates that the negative costimulatory signal can inhibit the anti-tuberculosis immunity of neutrophils, which may open up a new way for clinical treatment of tuberculosis.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R378.911

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相關(guān)期刊論文 前1條

1 姜麗娜;姚春艷;金齊力;賀文欣;李柏青;;IL-12增強結(jié)核病患者中性粒細胞吞噬和殺傷結(jié)核桿菌的活性[J];細胞與分子免疫學雜志;2011年11期

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