【摘要】：肺結核是長期嚴重危害人類健康的慢性傳染病，也是單因素所致感染性疾病中病死率最高的疾病之一。結核性胸膜炎作為肺外結核的類型之一，約占肺結核患者的10%-20%，其中約10%-30%的結核性胸膜炎患者臨床有胸腔積液表現(xiàn)。結核性胸腔積液作為常見的滲出性胸腔積液之一，胸水Th1細胞介導的免疫反應起主導作用，但近來研究結果表明，除了Th1型免疫反應，Th2、Th9、Th17、Th22等免疫細胞亞群及協(xié)同刺激分子在結核性胸腔積液的發(fā)生、發(fā)展中亦具有重要的作用。研究發(fā)現(xiàn)，協(xié)同刺激分子PD-L1（programmed death-1ligand1，程序性死亡配體1，又名B7-H1）在激活的T淋巴細胞、B淋巴細胞及單核-巨噬細胞等多種免疫細胞表面表達增高，并與機體的免疫抑制相關。業(yè)已證明，肺結核患者外周血中PD-1、PD-L1表達顯著增高，可能與結核病免疫耐受及慢性化炎癥相關。許多協(xié)同刺激分子如OX40、OX40L（OX40配體）、B7-H3和CTLA-4（細胞毒T淋巴細胞相關抗原-4）等除膜型外，還以可溶性的形式存在。既往對肺癌患者外周血可溶性PD-L1（sPD-L1）檢測發(fā)現(xiàn)，肺癌患者血清sPD-L1表達異常增高，并與腫瘤分期、轉移和療效相關。而胸腔積液中是否存在sPD-L1及sPD-L1在結核性胸腔積液中的生物學意義的研究并不多見。 本研究中，我們采用蘇州大學生物技術研究所自主研發(fā)的特異性人sPD-L1的酶聯(lián)免疫吸附法（ELISA）檢測體系對結核性胸腔積液患者開展深入研究，檢測結核性胸腔積液中sPD-L1的表達，分析其臨床指導意義，揭示sPD-L1和PD-1/PD-L1協(xié)同刺激信號與結核性胸腔積液疾病發(fā)生、發(fā)展的關系，進一步探究sPD-L1在結核性胸腔積液中的生物學意義。為免疫學方法在結核性胸腔積液中的治療提供依據(jù)。 一、可溶性PD-L1在結核性胸膜炎患者胸腔積液和外周血中的表達水平及臨床意義 【目的】檢測結核性胸膜炎患者胸腔積液和外周血中可溶性PD-L1的表達水平并探討其臨床意義。 【方法】篩選2012年6月至2013年3月蘇州大學附屬第二醫(yī)院呼吸科收治的初診胸腔積液患者68例，其中結核性胸腔積液組共24例，惡性胸腔積液組共30例，非結核、非惡性胸腔積液組14例。收集上述人群胸腔積液和外周血清并記錄其臨床資料。采用酶聯(lián)免疫吸附方法（ELISA）檢測胸腔積液和外周血中sPD-L1的水平。流式細胞法（FCM）檢測胸腔積液和外周血中CD4+T細胞的表達、CD8+T細胞的表達、PD-1在CD4+T細胞上的表達（CD4+-PD-1）、PD-1在CD8+T細胞上的表達（CD8+-PD-1）、CD14+單核細胞和PD-L1在CD14+單核細胞上的表達（CD14+-PD-L1）。反轉錄PCR法檢測胸腔積液中PD-L1、基質金屬蛋白酶3（MMP-3）基因表達。受試者工作特征（ROC）曲線分析sPD-L1對結核性胸腔積液的診斷價值。 【結果】結核性胸腔積液中sPD-L1含量顯著高于惡性組和非結核、非惡性組（P0.0001）；所有患者胸腔積液中的sPD-L1平均含量顯著高于外周血中（P=0.0033）。結核組胸腔積液中CD8+細胞比例和PD-L1在CD14+單核細胞表達（CD14+-PD-L1）比例均高于惡性組及非結核、非惡性組（P=0.0001，P0.0001）。反轉錄PCR結果顯示結核性胸腔積液中PD-L1mRNA表達增高，且與MMP-3表達水平相關（r=0.887，P0.0001）。ROC曲線分析顯示，單因素檢測胸腔積液sPD-L1診斷結核性胸腔積液的敏感度為82.6%，特異度為82.9%，曲線下面積為0.840。 【結論】 sPD-L1反映了不同病因胸腔積液微環(huán)境中不同的免疫功能狀態(tài)，結核性胸腔積液中sPD-L1表達明顯增高，可能與結核性胸腔積液的發(fā)生、發(fā)展相關，胸水中sPD-L1檢測有助于結核性胸腔積液的鑒別診斷。 二、結核性胸腔積液中IFN-γ和IFN-的表達及PD-1/PD-L1協(xié)同刺激信號在結核性胸膜炎中的免疫學作用 【目的】檢測結核性胸腔積液中IFN-γ和IFN-的表達水平并分析其臨床意義；探討PD-1/PD-L1協(xié)同刺激信號在結核性胸膜炎中的免疫學作用。 【方法】收集上述68例胸腔積液患者胸腔積液標本，采用ELISA法檢測胸腔積液中IFN-γ和IFN-的表達水平。分離健康成人外周血單個核細胞（PBMCs），以不同濃度結核性胸腔積液刺激培養(yǎng)后采用流式細胞術（FCM）檢測PBMCs細胞表面PD-L1的表達變化，CCK-8摻入法檢測PBMCs增殖的改變。免疫磁珠法分選結核性胸腔積液中T淋巴細胞和CD14+單核細胞，CCK-8摻入法檢測共培養(yǎng)體系中T細胞增殖的改變。 【結果】結核性胸腔積液組中IFN-γ含量顯著高于惡性組和非結核、非惡性組（P0.0001）；而三組患者胸腔積液中IFN-含量無統(tǒng)計學差異（P0.05）。胸腔積液中sPD-L1表達與Th1型主要細胞因子IFN-γ及胸腔積液腺苷脫氨酶（ADA）表達成正相關（P=0.0004，P0.0001）。聯(lián)合胸腔積液sPD-L1、CD14+-PD-L1、IFN-γ及ADA可使診斷結核性胸腔積液的敏感度達87.0%，特異度達100%，，曲線下面積達0.981。結核性胸腔積液可體外刺激CD14+單核細胞表面PD-L1的表達及PBMCs的增殖。高表達PD-L1的胸腔積液單核細胞能通過PD-1/PD-L1信號抑制T淋巴細胞的增殖和活化，采用抗PD-L1抗體特異性阻斷該抑制信號可部分恢復T淋巴細胞的增殖活性（P=0.005）。 【結論】結核性胸腔積液中IFN-γ表達增高，與胸腔積液中sPD-L1表達成正相關；聯(lián)合IFN-γ及各臨床指標檢測有助于結核性胸腔積液的鑒別診斷；高表達的Th1型細胞因子IFN-γ可能與PD-1/PD-L1協(xié)同刺激信號在結核性胸膜炎中的免疫作用相關。 綜上所述，本課題研究獲得了以下研究結果：（1）結核性胸腔積液中sPD-L1、Th1型細胞因子IFN-γ、CD8+細胞表達和PD-L1在CD14+單核細胞表達增高，sPD-L1單因素檢測或聯(lián)合各臨床指標有助于結核性胸腔積液的診斷。（2）結核性胸腔積液可體外促進CD14+單核細胞表面PD-L1的表達及外周血單個核細胞的增殖，增高表達的PD-L1與T細胞表面PD-1受體相結合，抑制了機體的免疫效應，可能與結核性胸腔積液的發(fā)生、發(fā)展相關。（3）通過抗PD-L1單抗阻斷PD-1/PD-L1途徑可部分恢復T淋巴細胞增殖能力，為結核性胸腔積液的免疫治療提供了依據(jù)。
[Abstract]:Tuberculosis is a chronic infectious disease that seriously endangers human health for a long time. It is also one of the most fatal infectious diseases caused by single factor. As one of the common exudative pleural effusion, pleural effusion Th1 cell mediated immune response plays a leading role. However, recent studies have shown that in addition to Th1 immune response, immune cell subsets such as Th2, Th9, Th17, Th22 and costimulatory molecules also play an important role in the development of tuberculous pleural effusion. The expression of co-stimulatory molecule PD-L1 (programmed death-1 ligand 1, also known as B7-H1) on the surface of activated T lymphocytes, B lymphocytes and monocyte-macrophage and other immune cells was increased, which was related to the immunosuppression of the body. Many co-stimulatory molecules, such as OX40, OX40L (OX40 ligand), B7-H3 and CTLA-4 (cytotoxic T lymphocyte associated antigen-4), exist in soluble form besides membrane type. Previous detection of soluble PD-L1 (sPD-L1) in peripheral blood of lung cancer patients showed abnormal increase of sPD-L1 expression. The biological significance of sPD-L1 and sPD-L1 in tuberculous pleural effusion is rare.
In this study, we used a specific human sPD-L1 enzyme-linked immunosorbent assay (ELISA) system developed by the Institute of Biotechnology of Suzhou University to detect the expression of sPD-L1 in tuberculous pleural effusion, analyze its clinical significance, and reveal the synergistic stimulus letters of sPD-L1 and PD-1/PD-L1. To explore the biological significance of sPD-L1 in tuberculous pleural effusion and to provide evidence for immunological treatment of tuberculous pleural effusion.
1. Expression of soluble PD-L1 in pleural effusion and peripheral blood of patients with tuberculous pleurisy and its clinical significance
[Objective] To detect the expression of soluble PD-L1 in pleural effusion and peripheral blood of patients with tuberculous pleurisy and explore its clinical significance.
[Methods] From June 2012 to March 2013, 68 patients with newly diagnosed pleural effusion, including 24 tuberculous pleural effusion, 30 malignant pleural effusion, 14 non-tuberculous and non-malignant pleural effusion, were selected from the Department of Respiration, Second Affiliated Hospital of Suzhou University. The levels of sPD-L1 in pleural effusion and peripheral blood were detected by enzyme-linked immunosorbent assay (ELISA). The expressions of CD4+T cells, CD8+T cells, PD-1 on CD4+T cells, PD-1 on CD8+T cells, CD14+monocytes and PD-L1 on CD14+T cells were detected by flow cytometry (FCM). The expression of PD-L1 and MMP-3 in pleural effusion was detected by reverse transcription polymerase chain reaction (RT-PCR). The diagnostic value of sPD-L1 in tuberculous pleural effusion was analyzed by ROC curve.
[Results] The content of sPD-L1 in tuberculous pleural effusion was significantly higher than that in malignant and non-tuberculous pleural effusion and non-malignant pleural effusion (P 0.0001), and the average content of sPD-L1 in pleural effusion of all patients was significantly higher than that in peripheral blood (P = 0.0033). The results of RT-PCR showed that the expression of PD-L1 mRNA in tuberculous pleural effusion was increased and correlated with the expression of MMP-3 (r = 0.887, P 0.0001). ROC curve analysis showed that the sensitivity and specificity of single factor detection of pleural effusion sPD-L1 in the diagnosis of tuberculous pleural effusion were 82.6%, 82.9% and 82.9% respectively. The product is 0.840.
[Conclusion] The expression of sPD-L1 in tuberculous pleural effusion may be related to the occurrence and development of tuberculous pleural effusion. The detection of sPD-L1 in pleural effusion is helpful to the differential diagnosis of tuberculous pleural effusion.
2. The expression of IFN-gamma and IFN-in tuberculous pleural effusion and the immunological role of PD-1/PD-L1 costimulatory signal in tuberculous pleurisy
[Objective] To detect the expression of IFN-gamma and IFN-in tuberculous pleural effusion and analyze its clinical significance, and to explore the immunological role of PD-1/PD-L1 costimulatory signal in tuberculous pleurisy.
[Methods] The expression of IFN-gamma and IFN-in pleural effusion was detected by ELISA in 68 patients with pleural effusion. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy adults. The expression of PD-L1 on PBMCs was detected by flow cytometry (FCM) after stimulating culture with different concentrations of tuberculous pleural effusion. The proliferation of PBMCs was detected by CCK-8 incorporation. T lymphocytes and CD14+ monocytes in tuberculous pleural effusion were separated by immunomagnetic beads method and T cell proliferation was detected by CCK-8 incorporation method.
[Results] The levels of IFN-gamma in tuberculous pleural effusion group were significantly higher than those in malignant group and non-tuberculous and non-malignant group (P 0.0001), but there was no significant difference in IFN-content among the three groups (P 0.05). The expression of sPD-L1 in pleural effusion was positively correlated with the expression of IFN-gamma and adenosine deaminase (ADA) in pleural effusion (P = 0.0004, P = 0.0004). Combined with sPD-L1, CD14 +-PD-L1, IFN-gamma and ADA, the sensitivity, specificity and area under the curve of diagnosis of tuberculous pleural effusion were 87.0%, 100% and 0.981 respectively. Tuberculous pleural effusion could stimulate the expression of PD-L1 on the surface of CD14 + monocytes and the proliferation of PBMCs in vitro. D-L1 signal inhibited the proliferation and activation of T lymphocytes. Anti-PD-L1 antibody could partially restore the proliferative activity of T lymphocytes (P=0.005).
[Conclusion] The increased expression of IFN-gamma in tuberculous pleural effusion is positively correlated with the expression of sPD-L1 in pleural effusion, and the detection of IFN-gamma and its clinical parameters is helpful to the differential diagnosis of tuberculous pleural effusion, and the high expression of Th1 cytokine IFN-gamma may be the immunological phase of PD-1/PD-L1 co-stimulating signal in tuberculous pleurisy. Close.
To sum up, the following results were obtained: (1) The expression of sPD-L1, Th1 cytokines IFN-gamma, CD8 + cells and PD-L1 in CD14 + monocytes in tuberculous pleural effusion were increased. The detection of sPD-L1 single factor or the combination of clinical indicators were helpful to the diagnosis of tuberculous pleural effusion. (2) Tuberculous pleural effusion could promote the diagnosis of tuberculous pleural effusion in vitro. The expression of PD-L1 on the surface of D14+ monocytes and the proliferation of peripheral blood mononuclear cells. The increased expression of PD-L1 combined with the expression of PD-1 receptor on the surface of T cells inhibited the immune response of the body, which may be related to the occurrence and development of tuberculous pleural effusion. It provides a basis for immunotherapy of tuberculous pleural effusion.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R521.7

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