【摘要】：目的:肺癌是當(dāng)今死亡率最高的惡性腫瘤之一,近來研究發(fā)現(xiàn)肺組織慢性炎癥反應(yīng)與肺癌發(fā)生密切相關(guān)。在癌變開始階段,慢性炎癥反應(yīng)促進(jìn)肺免疫抑制微環(huán)境形成,即形成腫瘤相關(guān)炎癥微環(huán)境,促進(jìn)腫瘤的發(fā)生和發(fā)展。在腫瘤形成前期,它主要由骨髓衍生抑制細(xì)胞(myeloid-derived suppressor cell,MDSC),調(diào)節(jié)性T細(xì)胞(regulatory T cell,Treg),腫瘤相關(guān)巨噬細(xì)胞(tumor-associated macrophage,TAM),以及其分泌的細(xì)胞因子和趨化因子組成,抑制效應(yīng)T細(xì)胞的功能,使腫瘤細(xì)胞逃避免疫監(jiān)視功能,在腫瘤啟動(dòng)階段促進(jìn)肺癌發(fā)生;而在腫瘤形成后它還包括腫瘤細(xì)胞及其分泌的細(xì)胞因子,發(fā)揮免疫抑制作用,抑制抗腫瘤免疫,進(jìn)而促進(jìn)肺癌細(xì)胞的浸潤(rùn)和轉(zhuǎn)移。近年來大量文獻(xiàn)報(bào)道,許多惡性腫瘤(例如乳腺癌、結(jié)腸直腸癌、肺癌、腎癌、頭頸癌、膀胱癌、胃癌和卵巢癌)相關(guān)微環(huán)境中發(fā)現(xiàn)了腫瘤相關(guān)樹突狀細(xì)胞(tumor-associated dendritic cell,TADC)。與傳統(tǒng)樹突狀細(xì)胞(dendritic cell,DC)識(shí)別、遞呈抗原,通過抗原遞呈作用激活效應(yīng)性CD4~+T和細(xì)胞毒性CD8~+T細(xì)胞不同,TADC激活后不誘導(dǎo)效應(yīng)性T細(xì)胞活化,而是促進(jìn)具有負(fù)向免疫調(diào)控作用的Treg分化和擴(kuò)增,進(jìn)而發(fā)揮免疫抑制作用。其在細(xì)胞功能以及分子表型上與目前報(bào)道的具有免疫抑制作用的調(diào)節(jié)性DC(regulatory dendritic cell,DCreg)非常相似,如:上調(diào)CD11b,精氨酸酶I(arginase I),IDO,NO表達(dá),改變MHC-Ⅱ,CD86,CD80和CD11c等表型分子表達(dá);同時(shí)分泌炎癥抑制因子如TGF-β,IL-10和PGE-2等。目前對(duì)于TADC的研究多集中在腫瘤形成后,腫瘤細(xì)胞通過怎樣機(jī)制誘導(dǎo)TADC分化、浸潤(rùn),而腫瘤相關(guān)慢性炎癥反應(yīng)是否誘導(dǎo)DC發(fā)揮免疫抑制作用還不清楚。目前關(guān)于腫瘤相關(guān)炎癥介導(dǎo)肺癌發(fā)生的研究證實(shí):慢性炎癥反應(yīng)可以促進(jìn)MDSC和Treg分化和增值,進(jìn)而誘導(dǎo)肺免疫抑制微環(huán)境形成,促進(jìn)肺癌的發(fā)生。這一系列研究多采用麻醉藥烏拉坦誘導(dǎo)的細(xì)胞因子TNF-α依賴的炎癥介導(dǎo)的肺腺癌發(fā)生模型,抑制TNF-α依賴炎癥反應(yīng)可以減少M(fèi)DSC和Treg浸潤(rùn),進(jìn)而減少肺腺癌發(fā)生。因此本研究首先建立烏拉坦誘導(dǎo)的小鼠肺腺癌發(fā)生模型,觀察腫瘤相關(guān)DC(TADC)的浸潤(rùn)和表型改變情況,同時(shí)給予TNF-α中和抗體s TNFR:Fc抑制炎癥介導(dǎo)的肺腺癌發(fā)生,探討抑制炎癥反應(yīng)對(duì)TADC表型和功能的影響,評(píng)估TADC與Treg浸潤(rùn)的相關(guān)性。為進(jìn)一步揭示慢性炎癥是否誘導(dǎo)DC發(fā)揮免疫抑制作用,本實(shí)驗(yàn)在烏拉坦誘導(dǎo)肺組織炎癥期(腫瘤形成前期),給予s TNFR:Fc抑制TNF-α依賴的炎癥反應(yīng),探討對(duì)肺組織DC浸潤(rùn)、表型以及功能改變的影響。本研究旨在探討介導(dǎo)肺腺癌發(fā)生的TNF-α依賴的慢性炎癥反應(yīng)是否會(huì)誘導(dǎo)肺組織DC發(fā)揮免疫抑制功能,進(jìn)一步揭示DC在炎癥介導(dǎo)的肺腫瘤免疫抑制微環(huán)境中的作用和機(jī)制。方法:Balb/c小鼠腹腔注射烏拉坦每周一次,作為烏拉坦誘導(dǎo)的肺腺癌發(fā)生組;對(duì)照組小鼠給予PBS溶液腹腔注射每周一次。連續(xù)給予8周后停止處理,繼續(xù)飼養(yǎng)至半年后,建立烏拉坦誘導(dǎo)小鼠肺腺癌發(fā)生模型。小鼠處死后取肺組織標(biāo)本,觀察腫瘤結(jié)節(jié)形成情況,組織學(xué)切片觀察肺腺癌形成情況;免疫組化方法觀察Treg細(xì)胞浸潤(rùn)情況;FCM方法檢測(cè)TADC和Treg細(xì)胞浸潤(rùn)情況;FCM方法檢測(cè)TADC表型分子CD11c,CD11b,MHC-Ⅱ,CD80,CD86,CD274等表達(dá)情況。Balb/c小鼠腹腔注射烏拉坦每周一次,同時(shí)給予PBS溶液腹腔注射每周兩次,作為烏拉坦誘導(dǎo)的肺腺癌發(fā)生組;每周一次小鼠腹腔注射烏拉坦,同時(shí)給予每周兩次TNF-α中和抗體s TNFR:Fc,作為阻斷劑組;對(duì)照組小鼠給予PBS溶液腹腔注射每周三次。連續(xù)給予8周后停止處理,小鼠繼續(xù)飼養(yǎng)至半年后處死。計(jì)數(shù)小鼠肺組織表面腫瘤結(jié)節(jié)數(shù)目、組織學(xué)切片觀察肺腺癌形成情況,評(píng)估TNF-α中和抗體s TNFR:Fc對(duì)于肺腺癌形成的抑制作用;免疫組化方法觀察Treg細(xì)胞浸潤(rùn)情況;FCM方法檢測(cè)TADC和Treg細(xì)胞浸潤(rùn)情況;FCM方法檢測(cè)TADC表型分子CD11c,CD11b,MHC-Ⅱ,CD80,CD86,CD274等表達(dá)情況。體外分離純化肺組織DC與體外分離純化脾組織CD4~+T細(xì)胞共培養(yǎng),采用流式細(xì)胞術(shù)方法檢測(cè)DC對(duì)Treg的誘導(dǎo)作用。Balb/c小鼠腹腔注射烏拉坦每周一次,同時(shí)給予PBS溶液腹腔注射每周兩次,作為烏拉坦誘導(dǎo)的肺炎癥組;每周一次小鼠腹腔注射烏拉坦,同時(shí)給予每周兩次TNF-α中和抗體s TNFR:Fc,作為阻斷劑組;對(duì)照組小鼠給予PBS溶液腹腔注射每周三次。連續(xù)給予8周后處死小鼠。在烏拉坦誘導(dǎo)小鼠肺組織炎癥期(腫瘤形成前期),檢測(cè)肺組織中TNF-α,p-NF-κB,COX-2以及Treg細(xì)胞標(biāo)記物FOXP3的表達(dá),評(píng)估抑制TNF-α對(duì)肺組織炎癥反應(yīng)的影響。并觀察肺組織DC的浸潤(rùn)、表型改變以及功能變化,分析與Treg浸潤(rùn)的相關(guān)性,進(jìn)一步探討導(dǎo)致肺腺癌發(fā)生的TNF-α依賴的肺慢性炎癥反應(yīng)對(duì)DC免疫抑制作用的影響。結(jié)果:1烏拉坦誘導(dǎo)的炎癥相關(guān)肺腺癌中腫瘤相關(guān)DC(TADC)的浸潤(rùn)和表型改變情況1.1烏拉坦誘導(dǎo)炎癥相關(guān)肺腺癌形成免疫抑制微環(huán)境與對(duì)照組相比,給予烏拉坦腹腔注射組小鼠均出現(xiàn)肺腺癌,同時(shí)伴隨Treg細(xì)胞標(biāo)志性分子Foxp3的高表達(dá),提示Treg細(xì)胞浸潤(rùn)增加,誘導(dǎo)免疫抑制微環(huán)境的形成。1.2烏拉坦誘導(dǎo)的炎癥相關(guān)肺腺癌組織中TADC的浸潤(rùn)和表型分子的表達(dá)采用流式細(xì)胞學(xué)技術(shù),檢測(cè)小鼠肺臟組織中DCs浸潤(rùn)情況。與對(duì)照組相比,烏拉坦誘導(dǎo)肺腺癌組CD11c+B200-細(xì)胞以及CD11c+CD11b+細(xì)胞浸潤(rùn)比例及細(xì)胞數(shù)均顯著增加,同時(shí)伴有表型分子MHC-Ⅱ,CD11b和PD-L1表達(dá)增加。提示,免疫抑制微環(huán)境的形成可以誘導(dǎo)幼稚樹突狀細(xì)胞表型分子表達(dá)改變,可能發(fā)揮免疫抑制作用。1.3 TADC細(xì)胞因子分泌水平改變采用Real-time PCR檢測(cè)IL-1,IL-6,IL-10,IL-12,TNF-α,COX-2的相對(duì)表達(dá)量,結(jié)果顯示:與對(duì)照組相比,烏拉坦誘導(dǎo)肺腺癌組TADC中IL-6,IL-10,COX-2細(xì)胞因子表達(dá)明顯升高,而TNF-α表達(dá)減少。提示,烏拉坦誘導(dǎo)的炎癥相關(guān)肺腺癌中TADC細(xì)胞因子分泌能力發(fā)生改變,與細(xì)胞發(fā)揮免疫抑制作用密切相關(guān)。2抑制TNF-α依賴炎癥反應(yīng)對(duì)烏拉坦誘導(dǎo)的肺腺癌發(fā)生以及TADC浸潤(rùn)和表型分子表達(dá)的影響2.1給予TNF-α阻斷劑s TNFR:Fc對(duì)烏拉坦誘導(dǎo)肺腺癌發(fā)生的影響給予TNF-α阻斷劑s TNFR:Fc明顯抑制烏拉坦誘導(dǎo)的肺腺癌發(fā)生,肺表面腫瘤團(tuán)塊減少。HE形態(tài)學(xué)觀察TNF-α阻斷劑組低倍視野內(nèi)腫瘤團(tuán)塊數(shù)量明顯減少。提示TNF-α介導(dǎo)的慢性炎癥反應(yīng)在烏拉坦誘導(dǎo)肺腺癌發(fā)生中起關(guān)鍵作用。2.2抑制TNF-α介導(dǎo)的慢性炎癥反應(yīng)對(duì)肺腺癌組織中TADC以及Treg浸潤(rùn)的影響與烏拉坦誘導(dǎo)肺腺癌組比較,TNF-α阻斷劑組肺腺癌組織CD11c+B200-細(xì)胞、CD11c+CD274+細(xì)胞以及CD11c+CD11b+細(xì)胞浸潤(rùn)比例及細(xì)胞數(shù)均有明顯降低。采用FCM方法檢測(cè)CD4~+CD25~+Treg細(xì)胞浸潤(rùn),發(fā)現(xiàn)TNF-α阻斷劑明顯抑制肺腺癌組織中Treg細(xì)胞浸潤(rùn)。結(jié)果提示:TNF-α阻斷劑s TNFR:Fc抑制烏拉坦誘導(dǎo)的肺腺癌發(fā)生,肺腺癌中TADC和Treg的浸潤(rùn)減少。2.3抑制TNF-α介導(dǎo)的慢性炎癥反應(yīng)對(duì)肺腺癌組織中TADC表型分子表達(dá)的影響與烏拉坦誘導(dǎo)肺腺癌組比較,TNF-α阻斷劑組肺腺癌組織TADC表型分子CD80增加,而MHC-Ⅱ,CD11b和PD-L1表達(dá)下降;提示TNF-α相關(guān)炎癥與肺腺癌中TADC表型改變密切相關(guān)。3抑制TNF-α介導(dǎo)的慢性炎癥反應(yīng)對(duì)肺腺癌組織TADC誘導(dǎo)Treg擴(kuò)增的影響我們采用DC對(duì)Treg的誘導(dǎo)作用評(píng)估DC的免疫抑制功能。分別分離烏拉坦組肺腺癌和TNF-α阻斷劑組肺腺癌組織TADC與脾CD4~+T細(xì)胞共培養(yǎng),結(jié)果發(fā)現(xiàn)肺腺癌組織TADC明顯促進(jìn)CD4~+CD25~+Treg細(xì)胞擴(kuò)增;而給予阻斷劑抑制肺腺癌發(fā)生,其TADC誘導(dǎo)Treg細(xì)胞擴(kuò)增的能力減弱。提示慢性炎癥反應(yīng)誘導(dǎo)的肺腺癌中,TNF-α依賴的慢性炎癥與TADC發(fā)揮負(fù)向免疫調(diào)控作用密切相關(guān)。4導(dǎo)致肺腺癌發(fā)生的TNF-α依賴肺慢性炎癥反應(yīng)對(duì)DC免疫抑制作用的影響為進(jìn)一步揭示是否肺組織慢性炎癥誘導(dǎo)DC發(fā)揮免疫抑制作用,本實(shí)驗(yàn)在烏拉坦誘導(dǎo)肺組織炎癥期(腫瘤形成前期),給予TNF-α阻斷劑s TNFR:Fc抑制TNF-α依賴的炎癥反應(yīng),觀察DC浸潤(rùn)、表型以及功能改變情況。4.1抑制TNF-α對(duì)烏拉坦誘導(dǎo)小鼠肺組織炎癥反應(yīng)的影響形態(tài)學(xué)觀察發(fā)現(xiàn)烏拉坦明顯促進(jìn)肺泡上皮細(xì)胞TNF-α,p-NF-κB,COX-2蛋白表達(dá),提示給予烏拉坦處理8周可以誘導(dǎo)小鼠肺組織炎癥反應(yīng);與烏拉坦誘導(dǎo)的炎癥期肺組織相比,TNF-α阻斷劑組小鼠肺泡上皮細(xì)胞TNF-α,p-NF-κB,COX-2蛋白表達(dá)明顯減少,提示,TNF-α阻斷劑s TNFR:Fc可減輕烏拉坦誘導(dǎo)的肺組織炎癥反應(yīng)。4.2抑制TNF-α對(duì)烏拉坦誘導(dǎo)的炎癥期肺組織中Treg浸潤(rùn)的影響流式細(xì)胞術(shù)檢測(cè)發(fā)現(xiàn)烏拉坦明顯促進(jìn)小鼠肺組織CD4~+CD25~+Treg細(xì)胞浸潤(rùn);而與烏拉坦誘導(dǎo)的炎癥期肺組織比較,給予TNF-α阻斷劑s TNFR:Fc明顯抑制烏拉坦誘導(dǎo)的炎癥微環(huán)境中CD4~+CD25~+Treg細(xì)胞浸潤(rùn)。提示TNF-α依賴的肺炎癥反應(yīng)促進(jìn)免疫抑制微環(huán)境的形成。4.3 TNF-α依賴肺組織炎癥反應(yīng)中DCs浸潤(rùn)和表型分子表達(dá)情況烏拉坦誘導(dǎo)的肺組織炎癥反應(yīng)中,DC細(xì)胞浸潤(rùn)明顯增加,表型分子MHC-Ⅱ,CD11b和PD-L1表達(dá)增加,CD80表達(dá)下降。給予TNF-α阻斷劑s TNFR:Fc明顯抑制烏拉坦誘導(dǎo)的炎癥微環(huán)境中DC浸潤(rùn)與表型改變。提示TNF-α依賴的肺炎癥反應(yīng)促進(jìn)DC細(xì)胞浸潤(rùn),可能發(fā)揮免疫抑制功能。4.4 TNF-α依賴肺組織炎癥反應(yīng)中DC對(duì)Treg擴(kuò)增的影響分別分離烏拉坦誘導(dǎo)的炎癥期肺組織中和TNF-α阻斷劑組中DC與脾CD4~+T細(xì)胞共培養(yǎng),結(jié)果顯示烏拉坦誘導(dǎo)炎癥肺組織DC明顯促進(jìn)CD4~+CD25~+Treg細(xì)胞擴(kuò)增;而給予阻斷劑抑制炎癥反應(yīng),肺組織DC誘導(dǎo)Treg細(xì)胞擴(kuò)增的能力減弱。提示TNF-α依賴的慢性炎癥反應(yīng)在肺腺癌發(fā)生前期,即可誘導(dǎo)DC發(fā)揮負(fù)向免疫調(diào)控作用,誘導(dǎo)CD4~+CD25~+Treg細(xì)胞擴(kuò)增。結(jié)論:1烏拉坦誘導(dǎo)的肺腺癌組織中,Treg浸潤(rùn)增加,形成腫瘤相關(guān)炎癥免疫抑制微環(huán)境,其中腫瘤相關(guān)DC浸潤(rùn)增加,TADC表型分子及相關(guān)細(xì)胞因子表達(dá)發(fā)生改變,可能發(fā)揮免疫抑制作用。2給予TNF-α阻斷劑s TNFR:Fc降低了烏拉坦誘導(dǎo)的肺腺癌發(fā)生,減少肺腺癌組織中TADC和Treg的浸潤(rùn),逆轉(zhuǎn)了TADCs表型的改變,TADC誘導(dǎo)Treg細(xì)胞擴(kuò)增的能力減弱。提示TNF-α依賴的慢性炎癥反應(yīng)誘導(dǎo)肺腺癌發(fā)生時(shí),TADC發(fā)揮負(fù)向免疫調(diào)控作用,TNF-α/NF-κB介導(dǎo)炎癥反應(yīng)可能調(diào)控TADC功能改變。3在烏拉坦誘導(dǎo)的肺腺癌發(fā)生前期,烏拉坦誘導(dǎo)肺組織慢性炎癥反應(yīng),促進(jìn)DC細(xì)胞浸潤(rùn)以及表型改變。4給予TNF-α阻斷劑s TNFR:Fc抑制烏拉坦誘導(dǎo)炎癥反應(yīng),減少炎癥肺組織中Treg和DCs的浸潤(rùn)、逆轉(zhuǎn)DC表型分子改變,抑制炎癥肺組織中DCs對(duì)Treg的擴(kuò)增。提示導(dǎo)致肺腺癌發(fā)生的TNF-α依賴的慢性炎癥反應(yīng)可誘導(dǎo)DC發(fā)揮負(fù)向免疫調(diào)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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R734.2

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