【作者】 张健；

【导师】 华树成；

【作者基本信息】 吉林大学 ， 内科学， 2015， 博士

【摘要】 目的：结核性胸膜炎在肺外结核病中占有很大比例，在其所处的免疫微环境可观察到典型的由巨噬细胞诱导的迟发型超敏反应。巨噬细胞在机体抗结核分枝杆菌感染的免疫反应中发挥重要作用，它是机体抗结核杆菌感染的首要防线，同时亦是结核菌在宿主体内长期寄生的场所。巨噬细胞的功能状态与结核病的发生、发展及转归密切相关。目前，临床仍采用标准四联疗法治疗结核病，对药物的抗菌作用及机制研究已很深入及明确，然而药物对免疫细胞尤其对巨噬细胞的功能是否存在调节，目前研究并不深入。有限的研究仅停留在以动物实验、人巨噬细胞的体外实验、以及人外周血为观察对象的间接证据上，缺少针对人体结核病灶微环境进行的系统阐述。鉴于此，本研究拟通过对结核性胸膜炎患者经抗结核治疗后胸膜巨噬细胞表型、功能与基因型的检测，以及体外药物对胸膜巨噬细胞的直接刺激，观察胸膜巨噬细胞的上述变化，探讨四种抗结核药物对巨噬细胞产生的直接作用及调节机制，进而阐明巨噬细胞在结核病治疗中发挥的作用及机制，如能明确其作用靶点，可为结核性胸膜炎的治疗及转归提供重要的理论依据，对结核病化疗方案的调整、预后分析以及抗结核药物研发等方面均具有重要临床意义及价值。方法：流式细胞术检测胸膜巨噬细胞百分率的变化与其表面标志物的表达，以及胸膜巨噬细胞的吞噬功能；ELISA法检测抗结核治疗后胸腔积液中细胞因子的变化；对抗结核治疗前后的标本中的纯化巨噬细胞进行深度测序，qPCR对其进行验证；采用qPCR方法检测IS6110基因，对胸腔积液中结核菌进行绝对定量分析；抗结核药物单药及联合体外刺激纯化的巨噬细胞，流式细胞术检测巨噬细胞表面标志物表达，qPCR方法检测基因表达水平。结果：在体：1经抗结核药物治疗后，患者胸膜巨噬细胞表面标志物CD14、CD80、CD163及CD206荧光强度明显升高（MFI：p=0.045，p=0.026，p=0.016，p=0.005），后三种因子阳性细胞百分率亦增高(Percentage：p=0.041，p=0.006，p=0.003）;群体水平即非配对样本分析,与上述结果一致（MFI：p=0.017，p=0.0001，p<0.0001，p=0.0001; Percentage：p=0.002，p<0.0001，p=0.0003），CD86荧光强度升高（MFI：p=0.004）。2T细胞亚群CD3+CD4+及CD3+CD8+在治疗前后百分率略有降低，CD3-CD19+及CD3-CD56+仅个别样本有升高或降低，但均无统计学差异（p=0.578，p=0.424，p=0.602，p=0.923）, CD3+CD4+/CD3+CD8+亦无统计学意义（p=0.225）3同一样本经抗结核药物治疗前后，巨噬细胞吞噬多个Beads的细胞平均百分数从79.74±8.30%增至84.37±6.28%（p=0.035）。4抗结核治疗后胸腔积液中促炎因子IFN-γ及TNF-α水平均较治疗前降低（p<0.0001，p=0.045），抗炎因子IL-10水平亦降低（p<0.0001）。5经抗结核治疗后胸膜巨噬细胞有230个基因表达发生变化，其中156个基因表达上调，74个基因表达下调。与杀菌作用相关的基因COLEC12、CHI3L1、FNBP1L表达均上调（p=0.0363，0.0022，0.0034）；巨噬细胞产生的趋化因子CCL18及CXCL5表达增加（p=0.0024，0.0013）；促炎凋亡因子Caspase5表达量减少（p=0.0036）；而受干扰素调节的IFIT3、IFIT2及IFIT1表达均降低（p=0.0006，0.0015，0.0187）；趋化因子CXCL10及CCL8表达下调（p=0.0105，0.0155）。离体：6体外抗结核药物处理胸膜巨噬细胞，给药组巨噬细胞表面标志物CD14、CD80、CD86、CD163、CD206荧光强度不同程度增高，CD14、CD80、CD86、CD206的平均荧光强度以联合作用组药物作用最为明显（MFI: p=0.031, p=0.027, p=0.042, p=0.038）。7利福平或吡嗪酰胺单独处理细胞，均可增强CD80及CD86的表达（利福平MFI: p=0.041, p=0.024,吡嗪酰胺MFI: p=0.039,p=0.044）。8抗结核药物吡嗪酰胺或四种药物联合在Cmax剂量作用下分别使胸膜巨噬细胞CHI3L1mRNA表达上调（p=0.038，0.042）；异烟肼与吡嗪酰胺均可上调FNBP1L的表达，四药联合应用亦可使其表达升高（p=0.018，0.025，0.022）；吡嗪酰胺与四药联合应用上调CCL18的表达（p=0.036，0.045），吡嗪酰胺单药与四种药物联合作用于巨噬细胞的结果比较无差异。结论：1抗结核治疗结核性胸膜炎，促进病灶局部胸膜巨噬细胞的活化，对该处的淋巴细胞不产生明显作用。2抗结核药物联合应用，可直接刺激胸膜巨噬细胞表面标志物的表达升高，其中利福平与吡嗪酰胺发挥了重要作用。3抗结核药物联合应用，能直接增强胸膜巨噬细胞的吞噬功能，与吡嗪酰胺及异烟肼促CHI3L1或FNBP1L的基因表达有关。4吡嗪酰胺可以促进胸膜巨噬细胞趋化因子CCL18的表达上调，与结核性胸膜炎胸膜损伤后的修复有关。5在体抗结核药物联合应用，降低胸腔积液微环境中的促炎因子及抗炎因子水平，进而下调干扰素诱导基因IFIT的表达，可进一步抑制巨噬细胞对促炎因子的释放，使病灶微环境趋于稳态。6在体抗结核药物治疗可使结核性胸膜炎巨噬细胞在基因表达水平产生明显改变，与其参与的免疫防御反应密切相关，以杀菌作用相关基因的表达上调和干扰素介导的免疫反应相关因子的表达下调为主。更多还原

【Abstract】 Objective:Tuberculosis pleurily occupies a large proportion in theextrapulmonary tuberculosis,the immune microenvironment in its typicalby macrophages can be observed from inducing delayed-typehepersensitivity.Macrophages in the body’s immune response tomycobacterium tuberculosis infection plays an important role,it is thefirst line of the body resistance to mycobacterium tuberculosisinfection,at the same time parasitism in the host. State of themacrophages are closely associated with the occurrence,development andoutcome of tuberculosis.Currently,standard therapy to treat tuberculosisstill used in clinical,the antibacterial effect and mechanism research ofdrugs has been very deep and clear.However,especially for durgs on theimmune cells of macrophage function if there is a regulation,the presentstudy is not deep.Only limited research stay in animal experiments,invitro experiment,the macrophages of peripheral blood as observationobject on the indirect evidence of a lack of againt human tuberculosiskichen system in this paper,the microenvironment.In view of this,thisstudy proposed based on tuberculos pleurity patients after treatment withtuberculosis pleural macrophage phenotypes,fuction and genedetection,as well as the in vitro drug direct stimulus to the pleuralmacrophages,to observe the above changes of pleural macrophages,discusses four kinds of anti-tb drugs have direct effect on macrophages and regulation mechanism,thus illustrating the role of macrophages in TBtreatment and mechanism,such as to clatify.Its targets,for the treatment oftuberculous pleurisy and outcome provide important theoretical basis foranalysis of the adjustment and prognosis of chemotherapy fortuberculosis and so on research and development of anti-tb drugs hasimportant clinical significance and value.Method:FMC detect macrophages percentage changes and the expression ofsurface markers;FMC detecting the percentage of macrophages beingswallowed by bead;ELISA method to detect tuberculosis pleural effusionafter treatment in the change of cytokines;On the depth sequencing ofmacrophages that before and after treatment;QPCR method is used todetect gene1s6110absolute quantitative analysis was carried out on thepleural effusion of tuberculosis bacterium;Anti-tb drugs single-agent andjoint processing in ivtro purified macrophages and detect the geneexpression level of macrophages.Result:In vivo：1After treatment with anti-tb drugs,patients with pleural surfacecarkers CD14, CD80and CD163and CD206fluorescence intensityincreased significantly,after three factors positive cell percentage alsoincreased.(MFI:p=0.017,p=0.0001, p=0.0001;Percentage:p=0.002,p＜0.0001,p=0.0003),CD86fluorescence intensity alsoelevated(MFI:P=0.004).2After anti-tb treatment, The percentage of T cell subsetCD3+CD4+and CD3+CD8+both reduced. Individual sample only had an incease or lower in CD3-CD19+and CD3-CD56+,but neither of themhad statisticallydifferences(P=0.578,P=0.424,P=0.602,P=0.923),CD3+CD4+/CD3+CD8+had no statistically differences.3The same sample by anti-tb drugs before/after treatment,theaverage percentage of multiple beads of phagocyte cells from79.74±8.30%to84.37±6.28%.4The level of proinflammatory factory of IFN-gamma andTNF-alpha in the pleural effusion was to reduce after theatment.(p﹤0.0001,p=0.045);Anti-inflammatory factorys IL-10were also reduced.(p﹤0.0001).5There are230DEG of macrophages in two groups before/aftertreatment,in which156gene expression level up adjustment and74genedown adjustment.Gene associated with bactericidal action COLEC12,CH13L1,FNBP1L expression are raised (p=0.0363,0.0022,0.0363);Macrophages of chemokine CCL18and CXCL5expressionincreased(p=0.0024,0.0013);Factor caspase5proinflammatory apoptosisexpression to decrease(p=0.0036);By interferon responses IFIT3,IFIT2and IFIT1experession were lower(p=0.0006,0.0015,0.0006);ChemokinesCXCL10and CCL8expression(p=0.0105,0.0155).In vitro：6The fluorescence intensity of each marker in the groups that dealedwith drugs has diffirent degrees elevate,CD14,CD80,CD86,CD206average fluorescence intensity of drugs in combination groups in the mostobvious(MFI:P=0.031,P=0.027,P=0.042,P=0.038).7Either rifampicin or pyrazinamide individual deal with cells,canenhance the expression of CD80and CD86(MFI:p=0.041,p=0.024forR,p=0.039,p=0.044for Z). 8Pyrazinamide or four drugs in combination with the Cmax doserespectively under the pleural macrophages CH13L1,mRNA expressionupward(p=0.038,0.042);Isoniazid and pyrazinamide can increase theexpression of FNBP1L,four drugs in combination can also make itexpress elevated(p=0.018,0.025,0.022),Pyrazinamide and four drugs incombination can increase the espression of CCL18(P=0.036,0.045).Thereis no difference in the result of PZA and four drugs in combination actingon the macrophage.Conclusion:1The standard anti-tuberculosis drugs in the treatment of tuberculouspleurisy,promote local pleural macrophage activation,lymphoid cells donot produce obvious effect on it.2Anti-TB drugs combined application,can be directly stimulate thepleural surface marker expression of macrophages, rifampicin andpyrazinamide played an important role.3Joint application of anti-TB drugs can directly enhance the pleuramacrophage phagocytosis,INH and pyrazinamide can promote geneexpression of CH13L1or FNBP1L isoniazid.4Pyrazinamide can promote the pleura macrophage chemotacticfactor CCL18experssion,can be associated with tuberculous pleurisypleural damage after the repair.5Joint application of anti-TB drugs,reduce the proinflammatoryfactor in the pleural effusion microenvironment IFN-gamma,TNF alphaand anti-inflammatory factor level of IL-10,and then cut the expression ofIFIT,can further inhibit macrophage for the release of proinflammatoryfactor,lead to the steady state lesion microenvironment.6The anti-tuberculous treatment in vivito changed significantly ofthe gene expression levels of macrophages of tuberculous pleurisy and the chang is closely related to the participate in the immune defensereaction,its mainly related gene expression up of the sterilization functionand down of INF mediated immune respose.更多还原