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研究生:陳墨繁
研究生(外文):Mo-Fan Chen
論文名稱:IL-6經由Smad恢復TGF-beta對樹狀細胞成熟的抑制
論文名稱(外文):IL-6 Antagonizes the Inhibitory Activities of TGF-beta on Dendritic Cell Maturation via Smads
指導教授:朱瑞民朱瑞民引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:獸醫學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:51
中文關鍵詞:單核球來源樹突狀細胞犬傳染性花柳病SmadsIL-6TGF-beta
外文關鍵詞:Monocyte-derived-DCCTVTSmadsIL-6TGF-beta
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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
誌謝………………………………………………………………………………..…. .I
中文摘要………………………………………………………………………………II
Abstract ……………………………………………………………………………....III
Contents……………………………………………………………………………....IV
Abbriviation………………………………………………………………………….VI

Chapter 1. Background and Literature Review 1
1.1Dendritic cells 1
1.1.1 Subpopulations of dendritic cells 1
1.1.2 Surface molecule expression 2
1.1.3 Alterations in phenotype and functions caused by tumor 4
1.2 Canine transmissible venereal tumor 5
1.3 TGF-β 5
1.3.1 Effects of TGF-βin tumor immunology 6
1.3.2 TGF-β signaling 7
1.3.2.1 Smad-dependent pathway 7
1.3.2.2 Smad-independent pathway 10
1.3.2.3 Cross talk of Smads and other compounds 12
1.4 IL-6 ………………………………………………………………………..12
1.4.1 Pro tumoral activities of IL-6 13
1.4.2 Anti-tumoral activities of IL-6 13
1.4.3 Signaling pathway of IL-6 14
1.5 Objectives of this study 15
Chapter 2. Introduction 16
Chapter 3. Materials and Methods 19
3.1 Animals and generation of peripheral blood-derived DC 19
3.2 Flow cytometry analysis of DC phenotypes 19
3.3 Real-time RT-PCR 21
3.4 Allogeneic Mix Lymphocyte Reaction (MLR) 22
3.5 FITC-dextran uptake assay 22
3.6 Production of CTVT cell-culture supernatants 23
3.7 Cell fractionation and Western immunoblotting 23
3.8 Immunofluorescent staining for confocal microscopy 24
3.9 Enzyme-linked immunosorbant assay ( ELISA) 25
3.10 Statistical analysis 25
Chapter 4. Results 26
Chapter 1. Background and Literature Review 1
1.1Dendritic cells 1
1.1.1 Subpopulations of dendritic cells 1
1.1.2 Surface molecule expression 2
1.1.3 Alterations in phenotype and functions caused by tumor 4
1.2 Canine transmissible venereal tumor 5
1.3 TGF-β 5
1.3.1 Effects of TGF-βin tumor immunology 6
1.3.2 TGF-β signaling 7
1.3.2.1 Smad-dependent pathway 7
1.3.2.2 Smad-independent pathway 10
1.3.2.3 Cross talk of Smads and other compounds 12
1.4 IL-6 ………………………………………………………………………..12
1.4.1 Pro tumoral activities of IL-6 13
1.4.2 Anti-tumoral activities of IL-6 13
1.4.3 Signaling pathway of IL-6 14
1.5 Objectives of this study 15
Chapter 2. Introduction 16
Chapter 3. Materials and Methods 19
3.1 Animals and generation of peripheral blood-derived DC 19
3.2 Flow cytometry analysis of DC phenotypes 19
3.3 Real-time RT-PCR 21
3.4 Allogeneic Mix Lymphocyte Reaction (MLR) 22
3.5 FITC-dextran uptake assay 22
3.6 Production of CTVT cell-culture supernatants 23
3.7 Cell fractionation and Western immunoblotting 23
3.8 Immunofluorescent staining for confocal microscopy 24
3.9 Enzyme-linked immunosorbant assay ( ELISA) 25
3.10 Statistical analysis 25
Chapter 4. Results 26
4.1 TGF-β effects on DC 26
4.1.1 Phenotypic changes of canine DC 26
4.1.2 Inhibition of DC function 29
4.2 Recovery of TGF-β-suppressed activities 32
4.3 IL-6 effects on Smad2/3 TGF-β signaling 34
4.4 IL-6 effects on Smad2/3 nuclear translocation not by the induction of Smad7 ……………………………………………………………………….40
Chapter 5. Discussion 41
References 46
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