朱瑞民臺灣大學:獸醫學研究所陳墨繁Chen, Mo-FanMo-FanChen2010-05-042018-07-092010-05-042018-07-092009U0001-2001200914524900http://ntur.lib.ntu.edu.tw//handle/246246/178922在癌症病患，樹突狀細胞(DC)的功能明顯地受到抑制。而在犬傳染性花柳性的腫瘤(CTVT)模式中，當腫瘤進入自然的消退期，患犬之DC功能即恢復。已知CTVT會分泌高量的TGF-b.然而DC的功能缺失以及腫瘤進入消退期時之功能恢復，和TGF-b之間的關係以及機制上不明確。我們首先確認了CTVT分泌的TGF-b對單核球來原之C有抑制作用。給予TGF-b後，DC活化T淋巴的功能顯著的降低了，CD1a, CD40等分子的表現也減少了。以含有TGF-b之增長(P)期CTVT細胞上輕易培養DC, 也可以得到類似的效果。中和上清液中的TGF-b可以恢復DC活化T淋巴球的能力，也提高了DCMHCII的表現。實驗室之前的研究發現CTVT R期時，浸潤於腫瘤之間的淋巴球產生IL-6和TGF-b有很強的結抗效果。流是細胞儀的結果顯示IL-6可以有效的恢復DC被TGF-b抑制的MHCII表現。我們於是進一步測試IL-4是否會直接干擾TGF-b的傳導路徑。經由西方墨眼法以及共軛焦顯微鏡之觀察，我們發現IL-6會阻擋TGF-b引起之Smad2/3進入細胞核。在我們觀察IL-6減少核內Smad2/3的同時，並沒有偵測到抑制型Smad, Smad7表現而回饋控制TGF-b的傳導所致。本研究探討了宿主/癌症之間的交互關係，並提出力用IL-6回復癌症產生TGF-b 對 DC的傷害在癌症治療上的應用。The dendritic cell (DC) activities are significantly hampered in many cancers. It is interesting that, in a canine cancer model, canine transmissible venereal tumor (CTVT), when the cancer enters a spontaneous regression (R), the inhibited DC activities are restored. CTVT produces high levels of TGF-b However, the role of TGF-b and 5h3 mechanisms involved in the DC functional suppresion and the restoration is largely unknown. We confirmed that the CTVT-derived TGF-b suppressed monocyte-derivedDC activities. After TGF-b treatment, the T cell activation through DC was impeded and the supernatants from the progression phase CTVT that contained TGF-b. Neutralizing the TGF-b in the supernatants by specific monoclonal antibody reversed the inhibition of DC-induces lymphocyte stimulation and also enhanced the DC MHC II expression. Our precious sstudy indicated that IL-6 produces by tumor infiltrating lymphocytes in the CTVT R phase sxhibited strong anti-TGF-b activity. The recovery of the TGF-b inhibitrf DC activities by IL-6 was thereforer studied. The flow cytometry results showed a strong reaction of IL6 in restoring TGF0b0down-regulated MHC expression on DCs. We ffurther verified whether IL-6 interfered with the TGF-b activities directly. Using Western blotting and confocal microscopy, we found that the nuclear translocation of Smad2/3, a sign of signal transduction of TGF-b, was blocked by adding IL-6. The evidence that the Smad7, which is an inhibitory Smad, was not oncreased in expression by adding IL-6 indicated that the nuclear translocation of Smad2/3 blocked by IL-6 was not through Smad7 pathway. This study provides in depth understanding of the host/cancer interactions and possible applications of IL-6 to restore DC activities in cancers that produce TGF-b.口試委員審定書………………………………………………………………………I謝………………………………………………………………………………..…. .I文摘要………………………………………………………………………………IIbstract ……………………………………………………………………………....IIIontents……………………………………………………………………………....IVbbriviation………………………………………………………………………….VIhapter 1. Background and Literature Review 1.1Dendritic cells 1.1.1 Subpopulations of dendritic cells 1.1.2 Surface molecule expression 2.1.3 Alterations in phenotype and functions caused by tumor 4.2 Canine transmissible venereal tumor 5.3 TGF-β 5.3.1 Effects of TGF-βin tumor immunology 6.3.2 TGF-β signaling 7.3.2.1 Smad-dependent pathway 7.3.2.2 Smad-independent pathway 10.3.2.3 Cross talk of Smads and other compounds 12.4 IL-6 ………………………………………………………………………..12.4.1 Pro tumoral activities of IL-6 13.4.2 Anti-tumoral activities of IL-6 13.4.3 Signaling pathway of IL-6 14.5 Objectives of this study 15hapter 2. Introduction 16hapter 3. Materials and Methods 19.1 Animals and generation of peripheral blood-derived DC 19.2 Flow cytometry analysis of DC phenotypes 19.3 Real-time RT-PCR 21.4 Allogeneic Mix Lymphocyte Reaction (MLR) 22.5 FITC-dextran uptake assay 22.6 Production of CTVT cell-culture supernatants 23.7 Cell fractionation and Western immunoblotting 23.8 Immunofluorescent staining for confocal microscopy 24.9 Enzyme-linked immunosorbant assay ( ELISA) 25.10 Statistical analysis 25hapter 4. Results 26hapter 1. Background and Literature Review 1.1Dendritic cells 1.1.1 Subpopulations of dendritic cells 1.1.2 Surface molecule expression 2.1.3 Alterations in phenotype and functions caused by tumor 4.2 Canine transmissible venereal tumor 5.3 TGF-β 5.3.1 Effects of TGF-βin tumor immunology 6.3.2 TGF-β signaling 7.3.2.1 Smad-dependent pathway 7.3.2.2 Smad-independent pathway 10.3.2.3 Cross talk of Smads and other compounds 12.4 IL-6 ………………………………………………………………………..12.4.1 Pro tumoral activities of IL-6 13.4.2 Anti-tumoral activities of IL-6 13.4.3 Signaling pathway of IL-6 14.5 Objectives of this study 15hapter 2. Introduction 16hapter 3. Materials and Methods 19.1 Animals and generation of peripheral blood-derived DC 19.2 Flow cytometry analysis of DC phenotypes 19.3 Real-time RT-PCR 21.4 Allogeneic Mix Lymphocyte Reaction (MLR) 22.5 FITC-dextran uptake assay 22.6 Production of CTVT cell-culture supernatants 23.7 Cell fractionation and Western immunoblotting 23.8 Immunofluorescent staining for confocal microscopy 24.9 Enzyme-linked immunosorbant assay ( ELISA) 25.10 Statistical analysis 25hapter 4. Results 26.1 TGF-β effects on DC 26.1.1 Phenotypic changes of canine DC 26.1.2 Inhibition of DC function 29.2 Recovery of TGF-β-suppressed activities 32.3 IL-6 effects on Smad2/3 TGF-β signaling 34.4 IL-6 effects on Smad2/3 nuclear translocation not by the induction of Smad7 ……………………………………………………………………….40hapter 5. Discussion 41eferences 46application/pdf2212278 bytesapplication/pdfen-US單核球來源樹突狀細胞犬傳染性花柳病SmadsIL-6TGF-betaMonocyte-derived-DCCTVT[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/178922/1/ntu-98-R95629005-1.pdf