【作者】 唐颖；

【导师】 许力军；

【作者基本信息】 吉林大学 ， 内科学， 2014， 博士

【摘要】 背景：结核性胸腔积液和恶性胸腔积液是渗出性胸腔积液中最常见的两种类型。巨噬细胞是胸腔积液中的主要细胞组分，具有吞噬、清除异物和衰老伤亡细胞的功能，在机体的固有免疫应答中发挥了重要作用。巨噬细胞还是一种重要的抗原呈递细胞，可以有效的向T细胞呈递抗原，进而诱导T细胞的激活。根据巨噬细胞的功能巨噬细胞可以被分为两种极化类型：M1型和M2型。M1型主要起促炎、抗肿瘤的作用；而M2型主要起炎症修复和促进肿瘤生长的作用。巨噬细胞的不同活化状态是决定其在疾病中发挥作用的基础。巨噬细胞是机体抗结核免疫的第一道防线，结核分枝杆菌进入机体后能否存活或复制，与巨噬细胞的功能状态密切相关；另一方面,肿瘤相关的巨噬细胞可以通过释放许多趋化因子促进肿瘤生长和血管生成,并且可以降解基质促进肿瘤侵袭和转移。巨噬细胞在结核性和恶性胸腔积液患者胸腔局部免疫中到底发挥了什么作用还有待进一步研究。另外，尽管近年来肿瘤的诊疗技术取得了长足的进步，但对于良恶性胸腔积液的鉴别临床上还存在一定的困难。细胞学检测是区分良恶性胸腔积液的最主要的方法，然而胸水中过度增生的间皮细胞、组织细胞和退行性的间皮细胞通常显示特殊的形态学特征,使得肿瘤细胞难于识别。人类肺组织特异性基因Lunx在肺组织中特异表达，而在其他人类肿瘤及正常组织中极少表达或不表达，Lunx基因在快速鉴别浆膜腔积液的良恶性以及在判定浆膜腔积液中恶性肿瘤细胞的来源方面,可能具有一定的应用价值。因此本课题旨在明确结核性和恶性胸腔积液中巨噬细胞亚群的变化，为进一步研究巨噬细胞在二者的发生及发展中所发挥的作用提供理论基础；另外通过进一步检测胸腔积液中Lunx基因的表达水平，评价Lunx基因检测在恶性胸腔积液诊断中的作用及临床意义。目的：通过检测巨噬细胞及其亚型在结核性胸膜炎和恶性胸腔积液中的表达，探讨巨噬细胞在渗出性胸腔积液发病机制中的作用，进一步通过检测恶性胸腔积液中Lunx基因的表达，评估Lunx基因检测的价值及临床意义。方法：通过流式细胞技术检测结核性胸膜炎和恶性胸腔积液患者外周血、胸腔积液巨噬细胞亚型及其相关细胞因子的表达，采用微量样本多指标流式蛋白定量技术(CBA)和酶联免疫吸附试验(ELISA)检测TNF-, IL-1β, IL-6, IL-8, IL-10,IL-12和TGF-β细胞因子的含量，并分析其与临床指标之间的关系。采用实时定量RT-PCR检测胸水Lunx mRNA的表达水平。临床指标中血细胞计数、血沉、结核斑点实验、胸水酸碱度、白蛋白、乳酸脱氢酶、癌胚抗原、脱落细胞等均由检验科或病理科专科医生完成。结果：1.结核性胸膜炎患者外周血CD14+CD163-M1、CD14+CD163-IL-12+M1型巨噬细胞的数量较健康对照组明显增高，但低于相应的胸腔积液；增高的外周血CD14+CD163-IL-12+M1型巨噬细胞的数量与血清IL-12浓度呈明显正相关。2.结核性胸膜炎患者外周血CD14+CD163+M2、 CD14+CD163+CD206+M2、CD14+CD163+CD115+M2和CD14+CD163+CD206+CD115+M2型巨噬细胞的数量明显多于相应的胸腔积液。3.结核性胸膜炎患者外周血CD14+CD163-MAC387+M1和CD14+CD163-MAC387-M1型巨噬细胞的数量明显高于健康对照组，但是低于相应的胸腔积液。4.结核性胸膜炎患者血清IL-1, IL-6, IL-8, IL-12, TNF和TGF-的浓度明显高于健康对照组,但低于相应的胸腔积液，而血清IL-10浓度和健康对照组比较无统计学差异，但仍低于相应的胸腔积液水平。5.结核性胸膜炎患者血清TNF-和IL-12水平与ESR、 ESAT-6-IFN-γ-T、CFP-10-IFN-γ-T细胞数量呈明显正相关，而胸水中TNF-水平与ADA和LDH呈明显正相关。6.恶性胸腔积液患者外周血CD14+CD163+M2、CD14+CD163+IL-10+M2型巨噬细胞的数量较健康对照组明显增高，但低于相应的胸腔积液。外周血CD14+CD163-IL-12+M1型巨噬细胞的数量明显低于健康对照组，但高于相应的胸腔积液。7.恶性胸腔积液患者外周血CD14+CD163+M2、 CD14+CD163+CD206+M2、CD14+CD163+CD115+M2和CD14+CD163+CD206+CD115+M2型巨噬细胞的数量较健康对照组明显增高，但低于相应的胸腔积液。8.恶性胸腔积液患者外周血CD14+CD163+MAC387+M2和CD14+CD163+MAC387-M2型巨噬细胞的数量明显高于健康对照组，但是低于相应的胸腔积液。9.恶性胸腔积液患者血清IL-1, IL-6, IL-8, IL-10, TNF和TGF-的浓度明显高于健康对照组,但低于相应的胸腔积液，而血清IL-12浓度与健康对照组和相应的胸腔积液比较无统计学差异。10.恶性胸腔积液患者胸水TNF-水平与CEA、LDH呈明显正相关，而IL-10水平与CEA呈明显正相关。11. Lunx mRNA诊断肺癌所致恶性胸腔积液的特异性为95.9%，敏感性为84.9%，ROC曲线下面积为0.922，明显高于胸水脱落细胞和CEA的0.821和0.798。12. Lunx mRNA在肺特异性表达，所有Lunx mRNA阳性的恶性胸腔积液患者均被诊断为肺癌，而所有肺外肿瘤所致的恶性胸腔积液患者Lunx mRNA表达均为阴性，LunxmRNA对于肿瘤细胞来源的阳性预测值为100%。13.在CR和PR组, Lunx mRNA的表达水平在第1疗程化疗结束后明显下降，而在NC组没有明显变化，PD组则显著增加。14.在Lunx mRNA表达下降组，患者的中位生存时间是53周,而在Lunx mRNA表达上升组，患者的中位生存时间是25周，Lunx mRNA表达下降组的总生存期要明显长于Lunx mRNA表达上升组。结论：1.新发结核性胸膜炎患者体内巨噬细胞亚型以M1型为主，并且以胸膜腔局部表现更为明显，提示我们结核分枝杆菌感染诱发的促炎的M1型巨噬细胞反应参与了结核性胸膜炎的早期发病过程。2.恶性胸腔积液患者胸水中肿瘤相关巨噬细胞主要表现为M2型，进一步证实了M2型巨噬细胞在恶性胸腔积液发生与发展中的作用。3. Lunx mRNA可以作为诊断肺癌所致恶性胸腔积液的标志物之一，推荐临床广泛应用，监测恶性胸腔积液患者化疗前后Lunx mRNA表达水平的变化有助于临床医生预测患者对治疗的反应性，监测病情并进一步评估患者的预后。更多还原

【Abstract】 Background: Lung cancer and tuberculosis infection are the main factors for the formationof pleural effusion. Macrophages are infiltrating components in the pleural fluid. Macrophagescan phagocytize pathogen, scavenge the foreign sunstance and dead cells, and as a type ofantigen-presenting cells in the innate immune system, can efficiently present antigen to T cells intissue, which results in T cell activation. Macrophages acquire distinct functional phenotypes,depending on the microenvironment, and macrophages can be classically activated as M1oralternatively activated as M2. M1macrophages exert the anti-inflammatory and anti-tumor effect,while M2macrophages repair the inflammation and induce the growth of tumor. The differentactivation states of macrophages determine their role in different diseases. Macrophages are thefirst line to defend the mycobacterium tuberculosis (M.tb) infection. Whether the M. tb cansurvive or reproduce is closely related to the functional status of macrophages;on the other hand,tumor-associated macrophages (TAMs) promote tumor growth and angiogenesis via releasingmany chemokines, and induce the degradation of the matrix, and further promote tumor invasionand metastasis. The role of macrophages in patients with tuberculous (TPE) or malignant pleuraleffusion (MPE) in patients remains to be further studied. In addition, in recent years, althoughdiagnosis technology of cancer has made considerable progress, there are still some difficultiesfor the identification of benign and malignant pleural effusion. Cytology is the most importantmethod to distinguish between benign and malignant pleural effusion. However, hyperplasticmesothelial cells, rhagiocrine cells, and degenerative mesothelial cells often display specialmorphological characteristics in the pleural effusion, which makes it difficult to identify thetumor cells. Lunx is distinctively expressed in pulmonary carcinoma, with little or no expressionin other human tumors and normal tissues. The detection of Lunx mRNA maybe helpful for theidentification between benign and malignant effusion. Therefore, this study was to determine thechanges of macrophage subsets in TPEs and MPEs, and provide a theoretical basis for furtherresearch. In addition, we assess the clinical significance of Lunx mRNA detection in diagnosingMPEs caused by pulmonary carcinoma.Aim：The study is aimed to exam the role of different subsets of macrophages in pleural fluid (PF) and peripheral blood (PB) from patients with new onset TPEs and MPEs andinvestigating the role of macrophages in the pleural effusions, and further to assessing theclinical significance of Lunx mRNA detection in diagnosing MPEs caused by pulmonarycarcinoma.Methods：The numbers of PB and PF macrophage subsets in patients with new onset TPEand MPE were determined by flow cytometry. The concentrations of serum and PF cytokineswere determined by cytometric bead array (CBA) and enzyme-linked immunosorbentassay(ELISA). The potential association between the cytokines and the values of clinical measureswere analyzed. The levels of Lunx mRNA in the PF were determined by real-time PCR. Theclinical parameters including blood counts, erythrocyte sedimentation rate (ESR), T.SPOT.TB,PF Potential of hydrogen (PH), lactate dehydrogenase (LDH), glucose, albumin,carcino-embryonic antigen (CEA), cast-off and et al. were completed by the trained specialist.Results：1. The numbers of PB CD14+CD163-and CD14+CD163-IL-12+M1macrophagesin the patients with onset TPE were significantly greater than those in the HC, but less thanthose in the PF of the patients. The concentrations of serum IL-12were correlated positivelywith the numbers of PB CD14+CD163-IL-12+M1in the patients2. The numbers of CD14+CD163+, CD14+CD163+CD206+, CD14+CD163+CD115+,CD14+CD163+CD206+CD115+M2in the PB were significantly greater than those in the PF ofonset TPE patients.3. The numbers of PB CD14+CD163-MAC387+and CD14+CD163-MAC387-M1macrophages in the patients with onset TPE were significantly greater than those in the HC, butless than those in the PF of the patients.4. The concentrations of serum IL-1, IL-6, IL-8, IL-12, TNF, and TGF-in the onsetTPE patients were significantly higher than that in the HC, but lower than that in the PF of thepatients. In contrast, there was no significant difference in the levels of serum IL-10between theHC and patients, but the levels of serum IL-10were significantly lower than that in the PF ofpatients.5. The levels of serum TNF-and IL-12was correlated positively with the values of ESRand the numbers of ESAT-6-and CFP-10-specific IFN-γ secreting T cells in the onset TPEpatients. In addition, the levels of PF TNF-were correlated positively with the concentrationsof ADA and LDH in the patients.6. The numbers of PB CD14+CD163+and CD14+CD163+IL-10+M2macrophages in thepatients with MPE were significantly greater than those in the HC, but less than those in the PF of the patients. The numbers of PB CD14+CD163-IL-12+M1macrophages in the patients weresignificantly less than those in the HC, but greater than those in the PF of the patients.7. The numbers of CD14+CD163+, CD14+CD163+CD206+, CD14+CD163+CD115+,CD14+CD163+CD206+CD115+M2in the PB of MPE patients were significantly greater thanthose in the HC, but less than those in the PF of the patients.8. The numbers of PB CD14+CD163+MAC387+M2and CD14+CD163+MAC387-M2macrophages in the patients with MPE were significantly greater than those in the HC, but lessthan those in the PF of the patients.9. The concentrations of serum IL-1, IL-6, IL-8, IL-10, TNF, and TGF-in the MPEpatients were significantly higher than that in the HC, but lower than that in the PF of thepatients. However, there was no significant difference in the levels of serum IL-12between theHC and patients.10. The levels of PF TNF-and IL-12was correlated positively with the values of CEAand LDH in the MPE patients, and the levels of PF IL-10were correlated positively with theconcentrations of CEA in the patients11.The specificity and sensitivity of Lunx mRNA for the diagnosis of MPE caused bypulmonary carcinoma.were95.9%and84.9%. The area under the ROC curve was0.922, andhigher than0.821for cast-off cells or0.798for CEA.12. Lunx mRNA is distinctively expressed in pulmonary carcinoma. All of theLunx-positive patients with MPEs were diagnosed with pulmonary carcinoma, and allextrapulmonary carcinoma patients were Lunx-negative. The positive predictive value of LunxmRNA for the source of tumor cells was100%.13. Lunx mRNA expression for MPE patients caused by pulmonary carcinoma decreasedafter the first session of chemotherapy in the CR and PR groups, increased in the PD group,there was no change in the NC group14. The median overall survival for MPE patients caused by pulmonary carcinoma was53weeks in the decreased Lunx mRNA expression group, and it was25weeks in the increasedLunx mRNA expression group. The patients in the decreased Lunx mRNA expression grouphad longer overall survival times than those in the increased Lunx mRNA expression groupConclusions:1. M. tb infection induced a polarized pro-inflammatory M1response in thepatients with onset TPE, especially at the pathological site. The data indicate that the increasedM1responses contribute to the early pathogenesis of TPE in humans. 2. M2macrophages are predominant in TAMs in the PF of MPE patients, which furtherindicate the important role of M2macrophages in the pathogenesis of MPE.3.Lunx mRNA is an effective marker of pulmonary carcinoma, and should berecommended to use in the clinic. To monitor the Lunx mRNA expression levels may help thedoctor predict the response of patients to the chemotherapy, understand the stage of the diseases,and further assess the prognosis.更多还原