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系統識別號 U0026-1302201503481800
論文名稱(中文) 探討肺部樹突細胞於單層奈米碳管所誘發肺部纖維化中所扮演之角色
論文名稱(英文) The role of pulmonary dendritic cells in single walled carbon nanotubes induced pulmonary fibrosis
校院名稱 成功大學
系所名稱(中) 環境醫學研究所
系所名稱(英) Institute of Environmental and Occupational Health
學年度 103
學期 1
出版年 104
研究生(中文) 李孟蓁
研究生(英文) Meng-Chen Lee
學號 s76014073
學位類別 碩士
語文別 中文
論文頁數 46頁
口試委員 指導教授-張志欽
口試委員-謝奇璋
口試委員-王育民
中文關鍵字 樹突細胞  肺部纖維化  單層奈米碳管  輔助型T細胞  調節性T細胞 
英文關鍵字 Dendritic cells  pulmonary fibrosis  SWCNT  Th cells  Treg 
學科別分類
中文摘要 伴隨著奈米科技的蓬勃發展,奈米材料的應用也越來越廣泛,其中單層奈米碳管因高彈性、良好導電性的物質特性,已被廣泛應用於電子行業、電力儲存裝置、超導產品以及航空航太等。然而,奈米材料的吸入對人體所造成的潛在傷害以及其機制尚未釐清。根據先前動物研究指出,單層奈米碳管的暴露會引起小鼠肺部損傷、肉芽腫、上皮細胞間質轉化及纖維化的情形發生。樹突細胞為已知重要的抗原呈現細胞,將捕捉到的抗原呈現給T細胞,引發免疫反應。在本研究中,假設單層奈米碳管造成肺部損傷的微環境,誘導樹突細胞活化引發T細胞免疫反應及纖維化的發生。本篇研究主要去釐清單層奈米碳管暴露後,不同樹突細胞的生成變化和其對T細胞變化及肺纖維化產生的影響。使用C57BL6母鼠以口咽吸入方式暴露單層奈米碳管,暴露劑量為80 μg/mouse,分別至不同時間點(3天、1週、2週、4週、8週、12週)後犧牲。利用流式細胞儀分析定量3種不同類型之樹突細胞,包含CD11c+lowCD11b+MHCII+CD207+的蘭格漢氏樹突細胞、CD11c+highCD11b+lowMHCII+CD103+的發炎性CD103+樹突細胞以及CD11c+highCD11b+highMHCII+CD103- 的單核細胞衍生之樹突細胞進行定量分析。利用不同細胞標誌針對各種T淋巴細胞進行分型定量,包含第二型輔助T細胞(Th2; CD4+IL-4+)、第十七型輔助T細胞(Th17; CD4+IL17-A+)以及調節性T細胞(Treg; CD4+CD25+FoxP3+)。利用酵素連結免疫分析法量測CCL2、CCL19、CCL12、 CXCL21、IL17、GM-CSF以及TGF-β。此外,利用口服灌食方式給予已知樹突細胞抑制劑(VAG539),目的為進一步驗證樹突細胞在由單層奈米碳管所誘發之肺纖維化的貢獻。結果顯示肺部CCL2、CCL19、CCL12、 CXCL21、IL17、GM-CSF以及TGF-β在暴露單層奈米碳管第三天後濃度顯著上升。接著在流式細胞儀分析結果指出,暴露單層奈米碳管第三天後,三種不同型態的樹突細胞(包含CD11c+lowCD11b+MHCII+CD207+ 的蘭格漢氏樹突細胞、CD11c+highCD11b+lowMHCII+CD103+的發炎性CD103+樹突細胞以及CD11c+highCD11b+highMHCII+CD103- 的單核細胞衍生之樹突細胞)均顯著於肺部聚集,並分別於第二週及第四週時達到峰,第二型輔助T細胞於暴露單層奈米碳管第三天時顯著上升,第十七型輔助T細胞以及調節性T細胞於暴露一週時顯著上升,並均在暴露第四週時達到峰。在體外試驗的部分,小鼠暴露單層奈米碳管後,分離出脾臟細胞進行T細胞增生能力的分析,發現暴露單層奈米碳管一週後,體外脾臟T細胞在刀豆素(ConA)刺激下增生能力受到抑制,但在暴露單層奈米碳管兩週及三週T細胞增生並未受到影響。在加入VAG539處理後,發現三種不同型態的樹突細胞(包含CD11c+lowCD11b+MHCII+CD207+ 的蘭格漢氏樹突細胞、CD11c+highCD11b+lowMHCII+CD103+的發炎性CD103+樹突細胞以及CD11c+highCD11b+highMHCII+CD103- 的單核細胞衍生之樹突細胞)以及第二型輔助T細胞、第十七型輔助T細胞以及調節性輔助T細胞於暴露四週時數量顯著下降。羥脯氨酸測試實驗中,發現加入VAG539組別暴露四週時肺部羥脯氨酸含量顯著下降。
  綜合以上結果,本研究確認了樹突細胞於單層奈米碳管所誘發的肺部纖維化中的貢獻。
英文摘要 To investigate whether dendritic cells regulate the development of single walled carbon nanotube (SWCNT)-induced pulmonary fibrosis. Female C57BL6 mice were oropharyngeally aspirated with 80 μg SWCNT. The results show that CXCL12, CCL2, CCL19, CCL21, IL17, GM-CSF and TGF-β were significantly increased starting at 3 d post exposure to SWCNT. Flow cytometry analyses demonstrate that three different types of dendritic cells infiltrated the lung starting at 3 d, with Langerhans cells-derived dendritic cells (DCs) peaking at 2 wk, and inflammatory CD103+ DCs and monocytes-derived DCs at 4 wk. The number of Th2 in the lungs started to increase at 3 d post exposure to SWCNT, while Th17 and Treg significantly increased starting at 1 wk and peaked at 4 wk. Results ex vivo experiments show that splenic T cells proliferation index was temperaly decreased at 1 wk post exposure. Moreover, treatment with VAG539 attenuated the infiltration of dendritic cells, T cell immune respones and hydroxyproline contents at 4 wk post exposure. These findings indicate that lung DCs modulate SWCNT-induced pulmonary inflammation and fibrosis.
論文目次 中文摘要I
英文摘要III
致謝VI
目錄VII
圖目錄X
第一章序論
1.1前言1
1.2研究目的2
第二章文獻探討
2.1奈米科技與單層奈米碳管
2.1.1奈米科技概述3
2.1.2奈米碳管的歷史3
2.1.3單層奈米碳管的結構與物化特性3
2.2單層奈米碳管與健康效應
2.2.1肺纖維化概述4
2.2.2單層奈米碳管與肺部損傷4
2.3樹突細胞在生物體之功能
2.3.1樹突細胞的分布與表型5
2.3.2樹突細胞與免疫反應5
2.3.3樹突細胞與肺部損傷5
第三章研究材料與方法
3.1實驗設計架構7
3.2實驗材料
3.2.1實驗動物9
3.2.2單層奈米碳管製備9
3.3實驗方法
3.3.1小鼠暴露單層奈米碳管9
3.3.2樹突細胞抑制劑VAG539
3.3.3抽取老鼠支氣管肺泡沖洗液9
3.3.4小鼠肺臟單細胞懸浮液以及流式細胞儀分析9
3.3.5小鼠肺臟CD11c+細胞純化10
3.3.6酵素連結免疫分析法11
3.3.7羥脯氨酸 (hydroxyproline)測量12
3.3.8小鼠脾臟細胞體外培養12
3.3.9脾臟T細胞增生試驗12
3.3.10螢光免疫組織染色法13
3.3.11統計分析13
第四章結果
4.1肺部樹突細胞相關趨化激素與細胞激素時序性變化
4.1.1老鼠暴露單層奈米碳管後肺部均質液 CCL2、CCL19、CCL21以及CXCL12 表現量14
4.1.2小鼠肺組織均質液小鼠肺部纖維轉化蛋白TGF-β、GM-CSF以及IL-17表現量14
4.2單層奈米碳管暴露導致肺部樹突細胞浸潤
4.2.1蘭格漢氏樹突細胞於肺組織中時序性累積情形14
4.2.2發炎性CD103+樹突細胞於肺組織中時序性累積情形14
4.2.3單核細胞衍生之樹突細胞於肺組織中時序性累積情形15
4.2.4單層奈米碳管暴露之小鼠對樹突細胞於肺部浸潤的情形15
4.3單層奈米碳管暴露引發輔助型T細胞免疫反應
4.3.1第二型輔助T細胞於肺組織中時序性累積情形15
4.3.2第十七型輔助T細胞於肺組織中時序性累積情形15
4.3.3調節型T細胞於肺組織中時序性累積情形15
4.3.4單層奈米碳管暴露之小鼠對輔助T細胞於肺部浸潤的情形16
4.4暴露單層奈米碳管對全身性免疫之影響
4.4.1單層奈米碳管對脾臟T細胞增生能力之影響16
4.5樹突細胞的浸潤於單層奈米碳管所誘發的肺纖維化之貢獻
4.5.1樹突細胞抑制劑VAG539抑制劑於單層奈米碳管暴露之小鼠對肺部損傷之影響16
4.5.2樹突細胞抑制劑VAG539抑制劑於單層奈米碳管暴露之小鼠對蘭格漢氏樹突細胞的影響16
4.5.3樹突細胞抑制劑VAG539抑制劑於單層奈米碳管暴露之小鼠對發炎性樹突細胞的影響16
4.5.4樹突細胞抑制劑VAG53抑制劑於單層奈米碳管暴露之小鼠對單核細胞衍生樹突細胞的影響16
4.5.5樹突細胞抑制劑VAG539抑制劑於單層奈米碳管暴露之小鼠對樹突細胞於肺部浸潤的影響17
4.5.6樹突細胞抑制劑VAG539抑制劑於單層奈米碳管暴露之小鼠對第二型輔助T細胞的影響17
4.5.7樹突細胞抑制劑VAG539抑制劑於單層奈米碳管暴露之小鼠對第十七型輔助T細胞的影響17
4.5.8樹突細胞抑制劑VAG539抑制劑於單層奈米碳管暴露之小鼠對調節性輔助T細胞的影響17
4.5.9樹突細胞抑制劑VAG539抑制劑於單層奈米碳管暴露之小鼠對輔助型T細胞於肺部浸潤的影響17
第五章討論18
第六章結論22
第七章參考文獻23
圖表27
參考文獻 Annacker O, Coombes JL, Malmstrom V, Uhlig HH, Bourne T, Johansson-Lindbom B, et al. 2005. Essential role for CD103 in the T cell–mediated regulation of experimental colitis. The Journal of Experimental Medicine 202:1051-1061.
Ban M, Langonné I, Huguet N, Guichard Y, Goutet M. 2012. Iron oxide particles modulate the ovalbumin-induced Th2 immune response in mice. Toxicology Letters 216.
Bantsimba-Malanda C, Marchal-Sommé J, Goven D, Freynet O, Michel L, Crestani B, et al. 2010. A role for dendritic cells in bleomycin-induced pulmonary fibrosis in mice? American Journal of Respiratory and Critical Care Medicine 182:385-395.
Chang CC, Chen CY, Chiu HF, Dai SX, Liu MY, Yang CY. 2011. Elastases from inflammatory and dendritic cells mediate ultrafine carbon black induced acute lung destruction in mice. Inhalation Toxicology 23:616–626.
Chen BX, Wilson SR, M. Das DJC, Erlanger BF. 1998. Antigenicity of fullerenes: Antibodies specific for fullerenes and their characteristics. PNAS 95.
Chen Y, Haines CJ, Gutcher I, Hochweller K, Blumenschein WM, McClanahan T, et al. 2011. Foxp3+ regulatory T cells promote T helper 17 cell development in vivo through regulation of interleukin-2. Immunity 34:409-421.
Coombes J, Siddiqui K, Arancibia-Cárcamo C, Hall J, Sun C, Belkaid Y, et al. 2007. A functionally specialized population of mucosal cd103+ dcs induces foxp3+ regulatory T cells via a tgf-beta and retinoic acid-dependent mechanism. The Journal of Experimental Medicine 204:1757-1764.
Dermott RM, Ziylan U, Spehne D, Bausinger H, Lipske D, Mommaas M, et al. 2002. Birbeck granules are subdomains of endosomal recycling compartment in human epidermal langerhans cells, which form where langerin accumulates. Molecular Biology of the Cell 13:317-335.
Dhulst, Vermaelen, Brusselle, Joos, Pauwels. 2005. Time course of cigarette smoke-induced pulmonary inflammation in mice. European Respiratory Journal 26:204-213.
Gauldie J. 2002. Inflammatory mechanisms are a minor component of the pathogenesis of idiopathic pulmonary fibrosis. American Journal of Respiratory and Critical Care Medicine 165:1205-1206.
Grouard G, Rissoan M, Filgueira L, Durand I, Banchereau J, Liu Y. 1997. The enigmatic plasmacytoid T cells develop into dendritic cells with interleukin (IL)-3 and CD40-ligand . Journal of Experimental Medicine 185:1101-1111.
Hammerich L, Heymann F, Tacke F. 2010. Role of IL-17 and Th17 cells in liver diseases. Clinical and Developmental Immunology 2011.
Hauben E, Gregori S, Draghici E, Migliavacca B, Olivieri S, Woisetschläger M, et al. 2008. Activation of the aryl hydrocarbon receptor promotes allograft-specific tolerance through direct and dendritic cell-mediated effects on regulatory T cells. Blood 112:1214-1222.
Hautamaki RD, K D, Kobayashi, M R, Senior, D S, et al. 1997. Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science 277:2002-2004.
Hsieh WY, Chou CC, Ho CC, Yu SL, Chen HY, Chou H-YE, et al. 2012. Single-walled carbon nanotubes induce airway hyperreactivity and parenchymal injury in mice. American Journal of Respiratory Cell and Molecular Biology 46: 257–267.
Jaurand M-CF, Renier A, Daubriac J. 2009. Mesothelioma: Do asbestos and carbon nanotubes pose the same health risk? Particle and Fibre Toxicology 6:16
Kaspar S, Takemura T, Tschachler E, Ferrans V, Kaliner M, Shevach E. 1986. Dendritic cells with antigen-presenting capability reside in airway epithelium, lung parenchyma, and visceral pleura. Journal of Experimental Medicine 163:436-451.
Katwa P, Wang X, Urankar RN, Podila R, Hilderbrand SC, Fick RB, et al. 2012. A carbon nanotube toxicity paradigm driven by mast cells and the il-33/st2 axis. Nanotechnology 8.
Kryczek I, Banerjee M, Cheng P, Vatan L, Szeliga W, Wei S, et al. 2009. Phenotype, distribution, generation, and functional and clinical relevance of Th17 cells in the human tumor environments. Blood 114:1141-1149.
Lam C-W, James JT, McCluskey R, Hunter RL. 2004. Pulmonary toxicity of single-wall carbon nanotubes in mice 7 and 90 days after intratracheal instillation. Toxicological Sciences 77:126-134.
Lukacs N, Hogaboam C, Chensue S, Blease K, Kunkel S. 2001. Type 1/Type 2 cytokine paradigm and the progression of pulmonary fibrosis . Chest 120: 5S–8S.
Marchal-Sommé J, Uzunhan Y, Marchand-Adam S, Valeyre D, Soumelis V, Crestani B, et al. 2006. Cutting edge: Nonproliferating mature immune cells form a novel type of organized lymphoid structure in idiopathic pulmonary fibrosis. The Journal of Immunology 176:5735-5739.
Mellor AL, Munn DH. 2004. Ido expression by dendritic cells: Tolerance and tryptophan catabolism. Nature Reviews Immunology 4:762-774.
Mitchell L, Lauer F, Burchiel S, McDonald J. 2009. Mechanisms for how inhaled multiwalled carbon nanotubes suppress systemic immune function in mice. Nature Nanotechnology 4:451-456.
Miyahara Y, Odunsi K, Chen W, Peng G, Matsuzaki J, Wang R-F. 2007. Generation and regulation of human CD4+IL-17-producing T cells in ovarian cancer. Proceedings of the National Academy of Sciences 105:15505-15510.
Munn D, Sharma M, Lee J, Jhaver K, Johnson T, Keskin D, et al. 2002. Potential regulatory function of human dendritic cells expressing indoleamine 2,3-dioxygenase. Science 297:1867-1870.
Pacurari M, Yin X, Zhao J, Ding M, Leonard S, Schwegler-Berry D, et al. 2008. Raw single-wall carbon nanotubes induce oxidative stress and activate MAPKs, AP-1, NF-κb, and AKT in normal and malignant human mesothelial cells. Environ Health Perspect 116:1211–1217.
Park EJ, Roh J, Kim SN, Kang MS, Han YA, Kim Y, et al. 2011. A single intratracheal instillation of single-walled carbon nanotubes induced early lung fibrosis and subchronic tissue damage in mice. Organ Toxicity And MechaNnisms 85:1121–1131.
Parka EJ, Kimb H, Kimb Y, Yic J, Choid K, Parka K. 2009. Carbon fullerenes (c60s) can induce inflammatory responses in the lung of mice. Toxicology and Applied Pharmacology 244:226-233
Saetta M, Stefano AD, Turato G, Facchini F, Corbino L, Mapp C, et al. 1998. Cd8+ t-lymphocytes in peripheral airways of smokers with chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine 157:822-826.
Sakaguchi S. 2004. Naturally arising cd4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annual Review of Immunology 22:531-556.
Sallusto F, Lanzavecchia A. 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. The Journal of Experimental Medicine 179:1109-1118.
Selman M, Pardo A. 2006. Role of epithelial cells in idiopathic pulmonary fibrosis. Proceedings of the American Thoracic Society 3:364-372.
Shvedova AA, Kisin ER, Mercer R, Murray AR, Johnson VJ, Potapovich AI, et al. 2005. Unusual inflammatory and fibrogenic pulmonary responses to single-walled carbon nanotubes in mice. Am J Physiol Lung Cell Mol Physiol 289:L698 –L708.
Simonian P, Roark C, Wehrmann F, Lanham A, Valle FDd, Born W, et al. 2009. Th17-polarized immune response in a murine model of hypersensitivity pneumonitis and lung fibrosis. The Journal of Immunology 182:657-665.
Spits H, Couwenberg F, Bakker A, Weijer K, Uittenbogaart C. 2000. Id2 and id3 inhibit development of CD34+ stem cells into predendritic cell (pre-DC)2 but not into pre-dc1 evidence for a lymphoid origin of pre-dc2. J Exp Med 192:1775-1784.
Tkach AV, Shurin GV, Shurin MR, Kisin ER, Murray AR, Young SH, et al. 2011. Direct effects of carbon nanotubes on dendritic cells induce immune suppression upon pulmonary exposure. ACS Nano 5:5755–5762.
Vermaelen K, Pauwels R. 2005. Pulmonary dendritic cells. American Journal of Respiratory and Critical Care Medicine 172:530-551.
Wang X, Katwa P, Podila R, Chen P, Ke PC, Rao AM, et al. 2011. Multi-walled carbon nanotube instillation impairs pulmonary function in C57BL/6 mice. Particle and Fibre Toxicology 8.
Wang X, Podila R, Shannahan JH, Rao AM, Brown JM. 2013. Intravenously delivered graphene nanosheets and multiwalled carbon nanotubes induce site-specific Th2 inflammatory responses via the il-33/st2 axis. International Journal of Nanomedicine 8.
Warhei DB, Laurence BR, Reed KL, Roach DH, Reynolds GAM, Webb TR. 2004. Comparative pulmonary toxicity assessment of single-wall carbon nanotubes in rats. Toxicol Science 77:117-125.
Yang H, Liu C, Yang D, Zhang H, Xi Z. 2008. Comparative study of cytotoxicity, oxidative stress and genotoxicity induced by four typical nanomaterials: The role of particle size, shape and composition. Journal of Applied Toxicology 29:69–78.

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