可溶性PD-L1分子在结核性胸腔积液中的表达特性及生物学意义
发布时间：2018-08-20 11:50
【摘要】：肺结核是长期严重危害人类健康的慢性传染病，也是单因素所致感染性疾病中病死率最高的疾病之一。结核性胸膜炎作为肺外结核的类型之一，约占肺结核患者的10%-20%，其中约10%-30%的结核性胸膜炎患者临床有胸腔积液表现。结核性胸腔积液作为常见的渗出性胸腔积液之一，胸水Th1细胞介导的免疫反应起主导作用，但近来研究结果表明，除了Th1型免疫反应，Th2、Th9、Th17、Th22等免疫细胞亚群及协同刺激分子在结核性胸腔积液的发生、发展中亦具有重要的作用。研究发现，协同刺激分子PD-L1（programmed death-1ligand1，程序性死亡配体1，又名B7-H1）在激活的T淋巴细胞、B淋巴细胞及单核-巨噬细胞等多种免疫细胞表面表达增高，并与机体的免疫抑制相关。业已证明，肺结核患者外周血中PD-1、PD-L1表达显著增高，可能与结核病免疫耐受及慢性化炎症相关。许多协同刺激分子如OX40、OX40L（OX40配体）、B7-H3和CTLA-4（细胞毒T淋巴细胞相关抗原-4）等除膜型外，还以可溶性的形式存在。既往对肺癌患者外周血可溶性PD-L1（sPD-L1）检测发现，肺癌患者血清sPD-L1表达异常增高，并与肿瘤分期、转移和疗效相关。而胸腔积液中是否存在sPD-L1及sPD-L1在结核性胸腔积液中的生物学意义的研究并不多见。 本研究中，我们采用苏州大学生物技术研究所自主研发的特异性人sPD-L1的酶联免疫吸附法（ELISA）检测体系对结核性胸腔积液患者开展深入研究，检测结核性胸腔积液中sPD-L1的表达，分析其临床指导意义，揭示sPD-L1和PD-1/PD-L1协同刺激信号与结核性胸腔积液疾病发生、发展的关系，进一步探究sPD-L1在结核性胸腔积液中的生物学意义。为免疫学方法在结核性胸腔积液中的治疗提供依据。 一、可溶性PD-L1在结核性胸膜炎患者胸腔积液和外周血中的表达水平及临床意义 【目的】检测结核性胸膜炎患者胸腔积液和外周血中可溶性PD-L1的表达水平并探讨其临床意义。 【方法】筛选2012年6月至2013年3月苏州大学附属第二医院呼吸科收治的初诊胸腔积液患者68例，其中结核性胸腔积液组共24例，恶性胸腔积液组共30例，非结核、非恶性胸腔积液组14例。收集上述人群胸腔积液和外周血清并记录其临床资料。采用酶联免疫吸附方法（ELISA）检测胸腔积液和外周血中sPD-L1的水平。流式细胞法（FCM）检测胸腔积液和外周血中CD4+T细胞的表达、CD8+T细胞的表达、PD-1在CD4+T细胞上的表达（CD4+-PD-1）、PD-1在CD8+T细胞上的表达（CD8+-PD-1）、CD14+单核细胞和PD-L1在CD14+单核细胞上的表达（CD14+-PD-L1）。反转录PCR法检测胸腔积液中PD-L1、基质金属蛋白酶3（MMP-3）基因表达。受试者工作特征（ROC）曲线分析sPD-L1对结核性胸腔积液的诊断价值。 【结果】结核性胸腔积液中sPD-L1含量显著高于恶性组和非结核、非恶性组（P0.0001）；所有患者胸腔积液中的sPD-L1平均含量显著高于外周血中（P=0.0033）。结核组胸腔积液中CD8+细胞比例和PD-L1在CD14+单核细胞表达（CD14+-PD-L1）比例均高于恶性组及非结核、非恶性组（P=0.0001，P0.0001）。反转录PCR结果显示结核性胸腔积液中PD-L1mRNA表达增高，且与MMP-3表达水平相关（r=0.887，P0.0001）。ROC曲线分析显示，单因素检测胸腔积液sPD-L1诊断结核性胸腔积液的敏感度为82.6%，特异度为82.9%，曲线下面积为0.840。 【结论】 sPD-L1反映了不同病因胸腔积液微环境中不同的免疫功能状态，结核性胸腔积液中sPD-L1表达明显增高，可能与结核性胸腔积液的发生、发展相关，胸水中sPD-L1检测有助于结核性胸腔积液的鉴别诊断。 二、结核性胸腔积液中IFN-γ和IFN-的表达及PD-1/PD-L1协同刺激信号在结核性胸膜炎中的免疫学作用 【目的】检测结核性胸腔积液中IFN-γ和IFN-的表达水平并分析其临床意义；探讨PD-1/PD-L1协同刺激信号在结核性胸膜炎中的免疫学作用。 【方法】收集上述68例胸腔积液患者胸腔积液标本，采用ELISA法检测胸腔积液中IFN-γ和IFN-的表达水平。分离健康成人外周血单个核细胞（PBMCs），以不同浓度结核性胸腔积液刺激培养后采用流式细胞术（FCM）检测PBMCs细胞表面PD-L1的表达变化，CCK-8掺入法检测PBMCs增殖的改变。免疫磁珠法分选结核性胸腔积液中T淋巴细胞和CD14+单核细胞，CCK-8掺入法检测共培养体系中T细胞增殖的改变。 【结果】结核性胸腔积液组中IFN-γ含量显著高于恶性组和非结核、非恶性组（P0.0001）；而三组患者胸腔积液中IFN-含量无统计学差异（P0.05）。胸腔积液中sPD-L1表达与Th1型主要细胞因子IFN-γ及胸腔积液腺苷脱氨酶（ADA）表达成正相关（P=0.0004，P0.0001）。联合胸腔积液sPD-L1、CD14+-PD-L1、IFN-γ及ADA可使诊断结核性胸腔积液的敏感度达87.0%，特异度达100%，，曲线下面积达0.981。结核性胸腔积液可体外刺激CD14+单核细胞表面PD-L1的表达及PBMCs的增殖。高表达PD-L1的胸腔积液单核细胞能通过PD-1/PD-L1信号抑制T淋巴细胞的增殖和活化，采用抗PD-L1抗体特异性阻断该抑制信号可部分恢复T淋巴细胞的增殖活性（P=0.005）。 【结论】结核性胸腔积液中IFN-γ表达增高，与胸腔积液中sPD-L1表达成正相关；联合IFN-γ及各临床指标检测有助于结核性胸腔积液的鉴别诊断；高表达的Th1型细胞因子IFN-γ可能与PD-1/PD-L1协同刺激信号在结核性胸膜炎中的免疫作用相关。 综上所述，本课题研究获得了以下研究结果：（1）结核性胸腔积液中sPD-L1、Th1型细胞因子IFN-γ、CD8+细胞表达和PD-L1在CD14+单核细胞表达增高，sPD-L1单因素检测或联合各临床指标有助于结核性胸腔积液的诊断。（2）结核性胸腔积液可体外促进CD14+单核细胞表面PD-L1的表达及外周血单个核细胞的增殖，增高表达的PD-L1与T细胞表面PD-1受体相结合，抑制了机体的免疫效应，可能与结核性胸腔积液的发生、发展相关。（3）通过抗PD-L1单抗阻断PD-1/PD-L1途径可部分恢复T淋巴细胞增殖能力，为结核性胸腔积液的免疫治疗提供了依据。
[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.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R521.7
本文编号：2193498
[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.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R521.7
【参考文献】
相关期刊论文 前2条
1 ;Identification of a novel splice variant of human PD-L1 mRNA encoding an isoform-lacking Igv-like domain[J];Acta Pharmacologica Sinica;2005年04期
2 Juan Ramón Larrubia;Selma Benito-Martínez;Joaquín Miquel;Miryam Calvino;Eduardo Sanz-de-Villalobos;Trinidad Parra-Cid;;Costimulatory molecule programmed death-1 in the cytotoxic response during chronic hepatitis C[J];World Journal of Gastroenterology;2009年41期
本文编号：2193498
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