TNF-α相关炎症介导的肺腺癌中树突状细胞免疫抑制功能的研究
发布时间：2018-09-10 11:42
【摘要】：目的:肺癌是当今死亡率最高的恶性肿瘤之一,近来研究发现肺组织慢性炎症反应与肺癌发生密切相关。在癌变开始阶段,慢性炎症反应促进肺免疫抑制微环境形成,即形成肿瘤相关炎症微环境,促进肿瘤的发生和发展。在肿瘤形成前期,它主要由骨髓衍生抑制细胞(myeloid-derived suppressor cell,MDSC),调节性T细胞(regulatory T cell,Treg),肿瘤相关巨噬细胞(tumor-associated macrophage,TAM),以及其分泌的细胞因子和趋化因子组成,抑制效应T细胞的功能,使肿瘤细胞逃避免疫监视功能,在肿瘤启动阶段促进肺癌发生;而在肿瘤形成后它还包括肿瘤细胞及其分泌的细胞因子,发挥免疫抑制作用,抑制抗肿瘤免疫,进而促进肺癌细胞的浸润和转移。近年来大量文献报道,许多恶性肿瘤(例如乳腺癌、结肠直肠癌、肺癌、肾癌、头颈癌、膀胱癌、胃癌和卵巢癌)相关微环境中发现了肿瘤相关树突状细胞(tumor-associated dendritic cell,TADC)。与传统树突状细胞(dendritic cell,DC)识别、递呈抗原,通过抗原递呈作用激活效应性CD4~+T和细胞毒性CD8~+T细胞不同,TADC激活后不诱导效应性T细胞活化,而是促进具有负向免疫调控作用的Treg分化和扩增,进而发挥免疫抑制作用。其在细胞功能以及分子表型上与目前报道的具有免疫抑制作用的调节性DC(regulatory dendritic cell,DCreg)非常相似,如:上调CD11b,精氨酸酶I(arginase I),IDO,NO表达,改变MHC-Ⅱ,CD86,CD80和CD11c等表型分子表达;同时分泌炎症抑制因子如TGF-β,IL-10和PGE-2等。目前对于TADC的研究多集中在肿瘤形成后,肿瘤细胞通过怎样机制诱导TADC分化、浸润,而肿瘤相关慢性炎症反应是否诱导DC发挥免疫抑制作用还不清楚。目前关于肿瘤相关炎症介导肺癌发生的研究证实:慢性炎症反应可以促进MDSC和Treg分化和增值,进而诱导肺免疫抑制微环境形成,促进肺癌的发生。这一系列研究多采用麻醉药乌拉坦诱导的细胞因子TNF-α依赖的炎症介导的肺腺癌发生模型,抑制TNF-α依赖炎症反应可以减少MDSC和Treg浸润,进而减少肺腺癌发生。因此本研究首先建立乌拉坦诱导的小鼠肺腺癌发生模型,观察肿瘤相关DC(TADC)的浸润和表型改变情况,同时给予TNF-α中和抗体s TNFR:Fc抑制炎症介导的肺腺癌发生,探讨抑制炎症反应对TADC表型和功能的影响,评估TADC与Treg浸润的相关性。为进一步揭示慢性炎症是否诱导DC发挥免疫抑制作用,本实验在乌拉坦诱导肺组织炎症期(肿瘤形成前期),给予s TNFR:Fc抑制TNF-α依赖的炎症反应,探讨对肺组织DC浸润、表型以及功能改变的影响。本研究旨在探讨介导肺腺癌发生的TNF-α依赖的慢性炎症反应是否会诱导肺组织DC发挥免疫抑制功能,进一步揭示DC在炎症介导的肺肿瘤免疫抑制微环境中的作用和机制。方法:Balb/c小鼠腹腔注射乌拉坦每周一次,作为乌拉坦诱导的肺腺癌发生组;对照组小鼠给予PBS溶液腹腔注射每周一次。连续给予8周后停止处理,继续饲养至半年后,建立乌拉坦诱导小鼠肺腺癌发生模型。小鼠处死后取肺组织标本,观察肿瘤结节形成情况,组织学切片观察肺腺癌形成情况;免疫组化方法观察Treg细胞浸润情况;FCM方法检测TADC和Treg细胞浸润情况;FCM方法检测TADC表型分子CD11c,CD11b,MHC-Ⅱ,CD80,CD86,CD274等表达情况。Balb/c小鼠腹腔注射乌拉坦每周一次,同时给予PBS溶液腹腔注射每周两次,作为乌拉坦诱导的肺腺癌发生组;每周一次小鼠腹腔注射乌拉坦,同时给予每周两次TNF-α中和抗体s TNFR:Fc,作为阻断剂组;对照组小鼠给予PBS溶液腹腔注射每周三次。连续给予8周后停止处理,小鼠继续饲养至半年后处死。计数小鼠肺组织表面肿瘤结节数目、组织学切片观察肺腺癌形成情况,评估TNF-α中和抗体s TNFR:Fc对于肺腺癌形成的抑制作用;免疫组化方法观察Treg细胞浸润情况;FCM方法检测TADC和Treg细胞浸润情况;FCM方法检测TADC表型分子CD11c,CD11b,MHC-Ⅱ,CD80,CD86,CD274等表达情况。体外分离纯化肺组织DC与体外分离纯化脾组织CD4~+T细胞共培养,采用流式细胞术方法检测DC对Treg的诱导作用。Balb/c小鼠腹腔注射乌拉坦每周一次,同时给予PBS溶液腹腔注射每周两次,作为乌拉坦诱导的肺炎症组;每周一次小鼠腹腔注射乌拉坦,同时给予每周两次TNF-α中和抗体s TNFR:Fc,作为阻断剂组;对照组小鼠给予PBS溶液腹腔注射每周三次。连续给予8周后处死小鼠。在乌拉坦诱导小鼠肺组织炎症期(肿瘤形成前期),检测肺组织中TNF-α,p-NF-κB,COX-2以及Treg细胞标记物FOXP3的表达,评估抑制TNF-α对肺组织炎症反应的影响。并观察肺组织DC的浸润、表型改变以及功能变化,分析与Treg浸润的相关性,进一步探讨导致肺腺癌发生的TNF-α依赖的肺慢性炎症反应对DC免疫抑制作用的影响。结果:1乌拉坦诱导的炎症相关肺腺癌中肿瘤相关DC(TADC)的浸润和表型改变情况1.1乌拉坦诱导炎症相关肺腺癌形成免疫抑制微环境与对照组相比,给予乌拉坦腹腔注射组小鼠均出现肺腺癌,同时伴随Treg细胞标志性分子Foxp3的高表达,提示Treg细胞浸润增加,诱导免疫抑制微环境的形成。1.2乌拉坦诱导的炎症相关肺腺癌组织中TADC的浸润和表型分子的表达采用流式细胞学技术,检测小鼠肺脏组织中DCs浸润情况。与对照组相比,乌拉坦诱导肺腺癌组CD11c+B200-细胞以及CD11c+CD11b+细胞浸润比例及细胞数均显著增加,同时伴有表型分子MHC-Ⅱ,CD11b和PD-L1表达增加。提示,免疫抑制微环境的形成可以诱导幼稚树突状细胞表型分子表达改变,可能发挥免疫抑制作用。1.3 TADC细胞因子分泌水平改变采用Real-time PCR检测IL-1,IL-6,IL-10,IL-12,TNF-α,COX-2的相对表达量,结果显示:与对照组相比,乌拉坦诱导肺腺癌组TADC中IL-6,IL-10,COX-2细胞因子表达明显升高,而TNF-α表达减少。提示,乌拉坦诱导的炎症相关肺腺癌中TADC细胞因子分泌能力发生改变,与细胞发挥免疫抑制作用密切相关。2抑制TNF-α依赖炎症反应对乌拉坦诱导的肺腺癌发生以及TADC浸润和表型分子表达的影响2.1给予TNF-α阻断剂s TNFR:Fc对乌拉坦诱导肺腺癌发生的影响给予TNF-α阻断剂s TNFR:Fc明显抑制乌拉坦诱导的肺腺癌发生,肺表面肿瘤团块减少。HE形态学观察TNF-α阻断剂组低倍视野内肿瘤团块数量明显减少。提示TNF-α介导的慢性炎症反应在乌拉坦诱导肺腺癌发生中起关键作用。2.2抑制TNF-α介导的慢性炎症反应对肺腺癌组织中TADC以及Treg浸润的影响与乌拉坦诱导肺腺癌组比较,TNF-α阻断剂组肺腺癌组织CD11c+B200-细胞、CD11c+CD274+细胞以及CD11c+CD11b+细胞浸润比例及细胞数均有明显降低。采用FCM方法检测CD4~+CD25~+Treg细胞浸润,发现TNF-α阻断剂明显抑制肺腺癌组织中Treg细胞浸润。结果提示:TNF-α阻断剂s TNFR:Fc抑制乌拉坦诱导的肺腺癌发生,肺腺癌中TADC和Treg的浸润减少。2.3抑制TNF-α介导的慢性炎症反应对肺腺癌组织中TADC表型分子表达的影响与乌拉坦诱导肺腺癌组比较,TNF-α阻断剂组肺腺癌组织TADC表型分子CD80增加,而MHC-Ⅱ,CD11b和PD-L1表达下降;提示TNF-α相关炎症与肺腺癌中TADC表型改变密切相关。3抑制TNF-α介导的慢性炎症反应对肺腺癌组织TADC诱导Treg扩增的影响我们采用DC对Treg的诱导作用评估DC的免疫抑制功能。分别分离乌拉坦组肺腺癌和TNF-α阻断剂组肺腺癌组织TADC与脾CD4~+T细胞共培养,结果发现肺腺癌组织TADC明显促进CD4~+CD25~+Treg细胞扩增;而给予阻断剂抑制肺腺癌发生,其TADC诱导Treg细胞扩增的能力减弱。提示慢性炎症反应诱导的肺腺癌中,TNF-α依赖的慢性炎症与TADC发挥负向免疫调控作用密切相关。4导致肺腺癌发生的TNF-α依赖肺慢性炎症反应对DC免疫抑制作用的影响为进一步揭示是否肺组织慢性炎症诱导DC发挥免疫抑制作用,本实验在乌拉坦诱导肺组织炎症期(肿瘤形成前期),给予TNF-α阻断剂s TNFR:Fc抑制TNF-α依赖的炎症反应,观察DC浸润、表型以及功能改变情况。4.1抑制TNF-α对乌拉坦诱导小鼠肺组织炎症反应的影响形态学观察发现乌拉坦明显促进肺泡上皮细胞TNF-α,p-NF-κB,COX-2蛋白表达,提示给予乌拉坦处理8周可以诱导小鼠肺组织炎症反应;与乌拉坦诱导的炎症期肺组织相比,TNF-α阻断剂组小鼠肺泡上皮细胞TNF-α,p-NF-κB,COX-2蛋白表达明显减少,提示,TNF-α阻断剂s TNFR:Fc可减轻乌拉坦诱导的肺组织炎症反应。4.2抑制TNF-α对乌拉坦诱导的炎症期肺组织中Treg浸润的影响流式细胞术检测发现乌拉坦明显促进小鼠肺组织CD4~+CD25~+Treg细胞浸润;而与乌拉坦诱导的炎症期肺组织比较,给予TNF-α阻断剂s TNFR:Fc明显抑制乌拉坦诱导的炎症微环境中CD4~+CD25~+Treg细胞浸润。提示TNF-α依赖的肺炎症反应促进免疫抑制微环境的形成。4.3 TNF-α依赖肺组织炎症反应中DCs浸润和表型分子表达情况乌拉坦诱导的肺组织炎症反应中,DC细胞浸润明显增加,表型分子MHC-Ⅱ,CD11b和PD-L1表达增加,CD80表达下降。给予TNF-α阻断剂s TNFR:Fc明显抑制乌拉坦诱导的炎症微环境中DC浸润与表型改变。提示TNF-α依赖的肺炎症反应促进DC细胞浸润,可能发挥免疫抑制功能。4.4 TNF-α依赖肺组织炎症反应中DC对Treg扩增的影响分别分离乌拉坦诱导的炎症期肺组织中和TNF-α阻断剂组中DC与脾CD4~+T细胞共培养,结果显示乌拉坦诱导炎症肺组织DC明显促进CD4~+CD25~+Treg细胞扩增;而给予阻断剂抑制炎症反应,肺组织DC诱导Treg细胞扩增的能力减弱。提示TNF-α依赖的慢性炎症反应在肺腺癌发生前期,即可诱导DC发挥负向免疫调控作用,诱导CD4~+CD25~+Treg细胞扩增。结论:1乌拉坦诱导的肺腺癌组织中,Treg浸润增加,形成肿瘤相关炎症免疫抑制微环境,其中肿瘤相关DC浸润增加,TADC表型分子及相关细胞因子表达发生改变,可能发挥免疫抑制作用。2给予TNF-α阻断剂s TNFR:Fc降低了乌拉坦诱导的肺腺癌发生,减少肺腺癌组织中TADC和Treg的浸润,逆转了TADCs表型的改变,TADC诱导Treg细胞扩增的能力减弱。提示TNF-α依赖的慢性炎症反应诱导肺腺癌发生时,TADC发挥负向免疫调控作用,TNF-α/NF-κB介导炎症反应可能调控TADC功能改变。3在乌拉坦诱导的肺腺癌发生前期,乌拉坦诱导肺组织慢性炎症反应,促进DC细胞浸润以及表型改变。4给予TNF-α阻断剂s TNFR:Fc抑制乌拉坦诱导炎症反应,减少炎症肺组织中Treg和DCs的浸润、逆转DC表型分子改变,抑制炎症肺组织中DCs对Treg的扩增。提示导致肺腺癌发生的TNF-α依赖的慢性炎症反应可诱导DC发挥负向免疫调控作用。
[Abstract]:Objective: Lung cancer is one of the malignant tumors with the highest mortality. Recently, studies have found that chronic inflammation of lung tissue is closely related to the occurrence of lung cancer. It is mainly composed of myeloid-derived suppressor cell (MDSC), regulatory T cell (Treg), tumor-associated macrophage (TAM), cytokines and chemokines secreted by the myeloid-derived suppressor cell (MDSC), tumor-associated macrophage (TAM), and suppresses the function of effector T cells to prevent tumor cells from escaping epidemic surveillance. Tumor initiation promotes the development of lung cancer; after tumor formation, it also includes tumor cells and cytokines secreted by tumor cells, which play an immunosuppressive role, inhibit anti-tumor immunity, and thus promote the infiltration and metastasis of lung cancer cells. In recent years, a large number of literatures have reported that many malignant tumors (such as breast cancer, colorectal cancer, lung cancer, kidney cancer, head and neck cancer). Tumor-associated dendritic cells (TADC) are found in cancer, bladder cancer, gastric cancer and ovarian cancer-related microenvironments. Recognizing and presenting antigens, TADC activates effector CD4~+T cells and cytotoxic CD8~+T cells through antigen presenting, but does not induce effector response after TADC activation. T cells activate, but promote the differentiation and amplification of Treg with negative immunomodulatory effect, and then exert immunosuppressive effect. It is very similar to regulatory dendritic cell (DCreg) with immunosuppressive effect in cell function and molecular phenotype, such as up-regulation of CD11b, arginase I, IDO, NO. The expression of MHC-II, CD86, CD80, CD11c and other phenotypic molecules are altered, and inflammatory inhibitors such as TGF-beta, IL-10, and PGE-2 are secreted. Current studies on tumor-associated inflammation-mediated lung cancer have shown that chronic inflammation can promote the differentiation and proliferation of MDSC and Treg, induce the formation of lung immunosuppressive microenvironment and promote the development of lung cancer. Inhibition of TNF-alpha dependent inflammation can reduce the infiltration of MDSC and Treg, and then decrease the occurrence of lung adenocarcinoma. In this study, we first established uratan-induced lung adenocarcinoma model in mice to observe the infiltration and phenotypic changes of tumor-associated DC (TADC), and at the same time, we gave TNF-alpha neutralizing antibody s TNFR:Fc to inhibit inflammation-mediated lung cancer. To investigate the effect of inhibiting inflammation on the phenotype and function of TADC and evaluate the correlation between TADC and Treg infiltration.To further reveal whether chronic inflammation can induce DC to exert immunosuppressive effect, this study was conducted to investigate the effect of s TNFR:Fc on TNF-alpha dependent inflammation in the inflammatory stage (precancerous stage) of lung tissue induced by uratan. The aim of this study was to investigate whether TNF-alpha-dependent chronic inflammation mediating the development of lung adenocarcinoma could induce lung DC to exert its immunosuppressive function, and to further explore the role and mechanism of DC in the inflammatory-mediated lung tumor immunosuppressive microenvironment. Methods: Balb/c mice were injected intraperitoneally. Uratan was injected intraperitoneally once a week into the control group and PBS solution was injected once a week. After 8 weeks of treatment, the model of lung adenocarcinoma induced by Uratan was established. Lung specimens were taken from mice to observe the formation of tumor nodules and tissues. The formation of lung adenocarcinoma was observed by sectioning; Treg cell infiltration was observed by immunohistochemistry; TADC and Treg cell infiltration were detected by FCM; TADC phenotype molecules CD11c, CD11b, MHC-II, CD80, CD86, CD274 were detected by FCM. Balb/c mice were intraperitoneally injected with urethane once a week and PBS solution was injected intraperitoneally twice a week. The mice in the control group were given PBS solution intraperitoneally three times a week. The mice in the control group were given PBS solution intraperitoneally three times a week. Number of tumor nodules on the tissue surface and histological sections were used to observe the formation of lung adenocarcinoma and evaluate the inhibitory effect of TNF-alpha neutralizing antibody s TNFR:Fc on lung adenocarcinoma formation; Treg cell infiltration was observed by immunohistochemical method; TADC and Treg cell infiltration were detected by FCM method; TADC phenotype molecules CD11c, CD11b, MHC-II, CD80, CD86, CD were detected by FCM method. The expression of 274 was detected by flow cytometry. Balb / c mice were intraperitoneally injected with urethane once a week, and PBS solution was injected intraperitoneally twice a week as urethane-induced pneumonia group. Uratan was injected intraperitoneally into mice, and TNF-a neutralizing antibody s TNFR:Fc was given twice a week as the blocker group; PBS solution was injected intraperitoneally three times a week in the control group. The mice were sacrificed after 8 weeks of continuous administration. The expression of FOXP3, a cell marker, was used to evaluate the effect of inhibiting TNF-alpha on lung inflammation. The infiltration, phenotypic and functional changes of pulmonary DC were observed. The correlation between Treg infiltration and the expression of FOXP3 was analyzed. The effect of TNF-alpha dependent chronic lung inflammation on DC immunosuppression was further investigated. Tumor-associated DC (TADC) infiltration and phenotypic changes in Tan-induced inflammatory-associated lung adenocarcinoma 1.1 Uratan-induced inflammatory-associated lung adenocarcinoma formation immunosuppressive microenvironment compared with the control group, Uratan-treated mice showed lung adenocarcinoma, accompanied by high expression of Treg cell marker Foxp3, suggesting Treg cells Infiltration of TADC and expression of phenotypic molecules in inflammation-associated lung adenocarcinoma tissues induced by urethane were detected by flow cytometry. Compared with the control group, urethane induced infiltration of CD11c+B200-cells and CD11c+CD11b+cells in lung adenocarcinoma tissues. The expression of phenotype molecules MHC-II, CD11b and PD-L1 was also increased. It was suggested that the formation of immunosuppressive microenvironment could induce the expression of phenotype molecules in immature dendritic cells, which might play an immunosuppressive role. 1.3 The secretion level of cytokines in TADC was detected by Real-time PCR for IL-1, IL-6, IL-10. Compared with the control group, the expression of IL-6, IL-10, COX-2 cytokines in TADC induced by urethane was significantly increased, while the expression of TNF-a was decreased. It suggested that the secretion of TADC cytokines in inflammatory-associated lung adenocarcinoma induced by urethane was altered and the cells exerted immunosuppressive effect. The effect of inhibiting TNF-alpha dependent inflammation on uratan-induced lung adenocarcinoma and TADC infiltration and phenotypic expression HE morphological observation showed that the number of tumor masses in low power visual field decreased significantly in TNF-alpha blocker group. It was suggested that chronic inflammatory reaction mediated by TNF-alpha played a key role in uratan-induced lung adenocarcinoma. 2.2 Inhibition of chronic inflammatory reaction mediated by TNF-alpha on TADC and Treg infiltration in lung adenocarcinoma tissue and uratan-induced lung adenocarcinoma group The infiltration ratio and number of CD11c+B200-cells, CD11c+CD274+ cells and CD11c+CD11b+ cells in lung adenocarcinoma tissue of TNF-alpha blocker group were significantly decreased. The infiltration of CD4~+CD25~+Treg cells was detected by FCM, and TNF-alpha blocker s TNFR:Fc was found to inhibit the infiltration of Treg cells in lung adenocarcinoma tissue. Inhibition of TADC and Treg infiltration in uratan-induced lung adenocarcinoma decreased. 2.3 Inhibition of TNF-alpha-mediated chronic inflammation on TADC phenotypic molecules expression in lung adenocarcinoma compared with uratan-induced lung adenocarcinoma increased TADC phenotypic molecules CD80, while MHC-II, CD11b and PD-L1 in TNF-alpha-blocker-induced lung adenocarcinoma. TNF-alpha-related inflammation is closely related to TADC phenotypic changes in lung adenocarcinoma. 3 Inhibition of TNF-alpha-mediated chronic inflammation on TADC-induced Treg amplification in lung adenocarcinoma tissue We assessed the immunosuppressive function of DCs by the induction of DCs on Treg. Uratan group and TNF-alpha blocker group were separated. Tissue TADCs were co-cultured with spleen CD4~+T cells. The results showed that TADCs significantly promoted the proliferation of CD4~+CD25~+Treg cells in lung adenocarcinoma, while blockers inhibited the occurrence of lung adenocarcinoma. The ability of TADCs to induce Treg cell proliferation was weakened. It suggested that TNF-alpha-dependent chronic inflammation and TADCs played a negative immunoregulatory role in lung adenocarcinoma induced by chronic inflammation. The effect of TNF-alpha-dependent chronic inflammation on DC immunosuppressive response in lung adenocarcinoma was studied. To further reveal whether chronic inflammation of lung tissue induces DC to exert immunosuppressive effect, TNF-alpha blockers s TNFR:Fc were administered to inhibit TNF-alpha dependence during the inflammatory stage (precancerous stage) of lung tissue induced by urethane. 4.1 Inhibition of TNF-alpha on inflammatory reaction in lung tissue induced by urethane showed that urethane significantly promoted the expression of TNF-alpha, p-NF-kappa B and COX-2 protein in alveolar epithelial cells, suggesting that urethane treatment for 8 weeks could induce inflammatory reaction in lung tissue of mice. The expression of TNF-alpha, p-NF-kappa B and COX-2 protein in alveolar epithelial cells of mice treated with TNF-alpha blocker was significantly lower than that in the inflammatory lung tissue induced by urethane, suggesting that TNF-alpha blocker s TNFR:Fc could attenuate the inflammatory response of lung tissue induced by urethane. 4.2 inhibited the effect of TNF-alpha on Treg infiltration in inflammatory lung tissue induced by urethane. Uratan significantly promoted the infiltration of CD4~+CD25~+Treg cells in lung tissues of mice, while TNF-alpha blocker s TNFR:Fc significantly inhibited the infiltration of CD4~+CD25~+Treg cells in inflammatory microenvironment induced by Uratan compared with lung tissues induced by Uratan. 4.3 The infiltration of DC s and the expression of phenotypic molecules in the inflammatory response of lung tissue depending on TNF-a increased infiltration of DC cells, increased expression of phenotypic molecules MHC-II, CD11b and PD-L1, and decreased expression of CD80 in the inflammatory response of lung tissue induced by urethane. Infiltration and phenotypic changes suggest that TNF-alpha-dependent pneumonia promotes infiltration of DC cells and may play an immunosuppressive role. 4.4 The effect of DC on Treg amplification in TNF-alpha-dependent pulmonary inflammation was separated and co-cultured with splenic CD4~+ T cells in the inflammatory lung tissue induced by uratan and TNF-alpha blocker groups, respectively. Tan-induced inflammation of lung tissue DC significantly promoted the proliferation of CD4~+CD25~+Treg cells, while blockers inhibited inflammation, and the ability of lung tissue DC to induce Treg cell proliferation was weakened. Conclusion: 1. Treg infiltration in lung adenocarcinoma tissues induced by urethane increases, forming tumor-associated inflammatory and immunosuppressive microenvironment, including tumor-associated DC.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
[Abstract]:Objective: Lung cancer is one of the malignant tumors with the highest mortality. Recently, studies have found that chronic inflammation of lung tissue is closely related to the occurrence of lung cancer. It is mainly composed of myeloid-derived suppressor cell (MDSC), regulatory T cell (Treg), tumor-associated macrophage (TAM), cytokines and chemokines secreted by the myeloid-derived suppressor cell (MDSC), tumor-associated macrophage (TAM), and suppresses the function of effector T cells to prevent tumor cells from escaping epidemic surveillance. Tumor initiation promotes the development of lung cancer; after tumor formation, it also includes tumor cells and cytokines secreted by tumor cells, which play an immunosuppressive role, inhibit anti-tumor immunity, and thus promote the infiltration and metastasis of lung cancer cells. In recent years, a large number of literatures have reported that many malignant tumors (such as breast cancer, colorectal cancer, lung cancer, kidney cancer, head and neck cancer). Tumor-associated dendritic cells (TADC) are found in cancer, bladder cancer, gastric cancer and ovarian cancer-related microenvironments. Recognizing and presenting antigens, TADC activates effector CD4~+T cells and cytotoxic CD8~+T cells through antigen presenting, but does not induce effector response after TADC activation. T cells activate, but promote the differentiation and amplification of Treg with negative immunomodulatory effect, and then exert immunosuppressive effect. It is very similar to regulatory dendritic cell (DCreg) with immunosuppressive effect in cell function and molecular phenotype, such as up-regulation of CD11b, arginase I, IDO, NO. The expression of MHC-II, CD86, CD80, CD11c and other phenotypic molecules are altered, and inflammatory inhibitors such as TGF-beta, IL-10, and PGE-2 are secreted. Current studies on tumor-associated inflammation-mediated lung cancer have shown that chronic inflammation can promote the differentiation and proliferation of MDSC and Treg, induce the formation of lung immunosuppressive microenvironment and promote the development of lung cancer. Inhibition of TNF-alpha dependent inflammation can reduce the infiltration of MDSC and Treg, and then decrease the occurrence of lung adenocarcinoma. In this study, we first established uratan-induced lung adenocarcinoma model in mice to observe the infiltration and phenotypic changes of tumor-associated DC (TADC), and at the same time, we gave TNF-alpha neutralizing antibody s TNFR:Fc to inhibit inflammation-mediated lung cancer. To investigate the effect of inhibiting inflammation on the phenotype and function of TADC and evaluate the correlation between TADC and Treg infiltration.To further reveal whether chronic inflammation can induce DC to exert immunosuppressive effect, this study was conducted to investigate the effect of s TNFR:Fc on TNF-alpha dependent inflammation in the inflammatory stage (precancerous stage) of lung tissue induced by uratan. The aim of this study was to investigate whether TNF-alpha-dependent chronic inflammation mediating the development of lung adenocarcinoma could induce lung DC to exert its immunosuppressive function, and to further explore the role and mechanism of DC in the inflammatory-mediated lung tumor immunosuppressive microenvironment. Methods: Balb/c mice were injected intraperitoneally. Uratan was injected intraperitoneally once a week into the control group and PBS solution was injected once a week. After 8 weeks of treatment, the model of lung adenocarcinoma induced by Uratan was established. Lung specimens were taken from mice to observe the formation of tumor nodules and tissues. The formation of lung adenocarcinoma was observed by sectioning; Treg cell infiltration was observed by immunohistochemistry; TADC and Treg cell infiltration were detected by FCM; TADC phenotype molecules CD11c, CD11b, MHC-II, CD80, CD86, CD274 were detected by FCM. Balb/c mice were intraperitoneally injected with urethane once a week and PBS solution was injected intraperitoneally twice a week. The mice in the control group were given PBS solution intraperitoneally three times a week. The mice in the control group were given PBS solution intraperitoneally three times a week. Number of tumor nodules on the tissue surface and histological sections were used to observe the formation of lung adenocarcinoma and evaluate the inhibitory effect of TNF-alpha neutralizing antibody s TNFR:Fc on lung adenocarcinoma formation; Treg cell infiltration was observed by immunohistochemical method; TADC and Treg cell infiltration were detected by FCM method; TADC phenotype molecules CD11c, CD11b, MHC-II, CD80, CD86, CD were detected by FCM method. The expression of 274 was detected by flow cytometry. Balb / c mice were intraperitoneally injected with urethane once a week, and PBS solution was injected intraperitoneally twice a week as urethane-induced pneumonia group. Uratan was injected intraperitoneally into mice, and TNF-a neutralizing antibody s TNFR:Fc was given twice a week as the blocker group; PBS solution was injected intraperitoneally three times a week in the control group. The mice were sacrificed after 8 weeks of continuous administration. The expression of FOXP3, a cell marker, was used to evaluate the effect of inhibiting TNF-alpha on lung inflammation. The infiltration, phenotypic and functional changes of pulmonary DC were observed. The correlation between Treg infiltration and the expression of FOXP3 was analyzed. The effect of TNF-alpha dependent chronic lung inflammation on DC immunosuppression was further investigated. Tumor-associated DC (TADC) infiltration and phenotypic changes in Tan-induced inflammatory-associated lung adenocarcinoma 1.1 Uratan-induced inflammatory-associated lung adenocarcinoma formation immunosuppressive microenvironment compared with the control group, Uratan-treated mice showed lung adenocarcinoma, accompanied by high expression of Treg cell marker Foxp3, suggesting Treg cells Infiltration of TADC and expression of phenotypic molecules in inflammation-associated lung adenocarcinoma tissues induced by urethane were detected by flow cytometry. Compared with the control group, urethane induced infiltration of CD11c+B200-cells and CD11c+CD11b+cells in lung adenocarcinoma tissues. The expression of phenotype molecules MHC-II, CD11b and PD-L1 was also increased. It was suggested that the formation of immunosuppressive microenvironment could induce the expression of phenotype molecules in immature dendritic cells, which might play an immunosuppressive role. 1.3 The secretion level of cytokines in TADC was detected by Real-time PCR for IL-1, IL-6, IL-10. Compared with the control group, the expression of IL-6, IL-10, COX-2 cytokines in TADC induced by urethane was significantly increased, while the expression of TNF-a was decreased. It suggested that the secretion of TADC cytokines in inflammatory-associated lung adenocarcinoma induced by urethane was altered and the cells exerted immunosuppressive effect. The effect of inhibiting TNF-alpha dependent inflammation on uratan-induced lung adenocarcinoma and TADC infiltration and phenotypic expression HE morphological observation showed that the number of tumor masses in low power visual field decreased significantly in TNF-alpha blocker group. It was suggested that chronic inflammatory reaction mediated by TNF-alpha played a key role in uratan-induced lung adenocarcinoma. 2.2 Inhibition of chronic inflammatory reaction mediated by TNF-alpha on TADC and Treg infiltration in lung adenocarcinoma tissue and uratan-induced lung adenocarcinoma group The infiltration ratio and number of CD11c+B200-cells, CD11c+CD274+ cells and CD11c+CD11b+ cells in lung adenocarcinoma tissue of TNF-alpha blocker group were significantly decreased. The infiltration of CD4~+CD25~+Treg cells was detected by FCM, and TNF-alpha blocker s TNFR:Fc was found to inhibit the infiltration of Treg cells in lung adenocarcinoma tissue. Inhibition of TADC and Treg infiltration in uratan-induced lung adenocarcinoma decreased. 2.3 Inhibition of TNF-alpha-mediated chronic inflammation on TADC phenotypic molecules expression in lung adenocarcinoma compared with uratan-induced lung adenocarcinoma increased TADC phenotypic molecules CD80, while MHC-II, CD11b and PD-L1 in TNF-alpha-blocker-induced lung adenocarcinoma. TNF-alpha-related inflammation is closely related to TADC phenotypic changes in lung adenocarcinoma. 3 Inhibition of TNF-alpha-mediated chronic inflammation on TADC-induced Treg amplification in lung adenocarcinoma tissue We assessed the immunosuppressive function of DCs by the induction of DCs on Treg. Uratan group and TNF-alpha blocker group were separated. Tissue TADCs were co-cultured with spleen CD4~+T cells. The results showed that TADCs significantly promoted the proliferation of CD4~+CD25~+Treg cells in lung adenocarcinoma, while blockers inhibited the occurrence of lung adenocarcinoma. The ability of TADCs to induce Treg cell proliferation was weakened. It suggested that TNF-alpha-dependent chronic inflammation and TADCs played a negative immunoregulatory role in lung adenocarcinoma induced by chronic inflammation. The effect of TNF-alpha-dependent chronic inflammation on DC immunosuppressive response in lung adenocarcinoma was studied. To further reveal whether chronic inflammation of lung tissue induces DC to exert immunosuppressive effect, TNF-alpha blockers s TNFR:Fc were administered to inhibit TNF-alpha dependence during the inflammatory stage (precancerous stage) of lung tissue induced by urethane. 4.1 Inhibition of TNF-alpha on inflammatory reaction in lung tissue induced by urethane showed that urethane significantly promoted the expression of TNF-alpha, p-NF-kappa B and COX-2 protein in alveolar epithelial cells, suggesting that urethane treatment for 8 weeks could induce inflammatory reaction in lung tissue of mice. The expression of TNF-alpha, p-NF-kappa B and COX-2 protein in alveolar epithelial cells of mice treated with TNF-alpha blocker was significantly lower than that in the inflammatory lung tissue induced by urethane, suggesting that TNF-alpha blocker s TNFR:Fc could attenuate the inflammatory response of lung tissue induced by urethane. 4.2 inhibited the effect of TNF-alpha on Treg infiltration in inflammatory lung tissue induced by urethane. Uratan significantly promoted the infiltration of CD4~+CD25~+Treg cells in lung tissues of mice, while TNF-alpha blocker s TNFR:Fc significantly inhibited the infiltration of CD4~+CD25~+Treg cells in inflammatory microenvironment induced by Uratan compared with lung tissues induced by Uratan. 4.3 The infiltration of DC s and the expression of phenotypic molecules in the inflammatory response of lung tissue depending on TNF-a increased infiltration of DC cells, increased expression of phenotypic molecules MHC-II, CD11b and PD-L1, and decreased expression of CD80 in the inflammatory response of lung tissue induced by urethane. Infiltration and phenotypic changes suggest that TNF-alpha-dependent pneumonia promotes infiltration of DC cells and may play an immunosuppressive role. 4.4 The effect of DC on Treg amplification in TNF-alpha-dependent pulmonary inflammation was separated and co-cultured with splenic CD4~+ T cells in the inflammatory lung tissue induced by uratan and TNF-alpha blocker groups, respectively. Tan-induced inflammation of lung tissue DC significantly promoted the proliferation of CD4~+CD25~+Treg cells, while blockers inhibited inflammation, and the ability of lung tissue DC to induce Treg cell proliferation was weakened. Conclusion: 1. Treg infiltration in lung adenocarcinoma tissues induced by urethane increases, forming tumor-associated inflammatory and immunosuppressive microenvironment, including tumor-associated DC.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
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