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系統識別號 U0026-2708201200393200
論文名稱(中文) 循環纖維細胞於單層奈米碳管誘發肺纖維化所扮演之角色
論文名稱(英文) The role of circulating fibrocytes 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 100
學期 2
出版年 101
研究生(中文) 陳慶瑜
研究生(英文) Chin-Yu Chen
電子信箱 virg7989@msn.com
學號 s76984082
學位類別 碩士
語文別 中文
論文頁數 72頁
口試委員 指導教授-張志欽
口試委員-劉明毅
口試委員-謝達斌
中文關鍵字 循環纖維細胞  肺纖維化  單層奈米碳管 
英文關鍵字 fibrocytes  pulmonary fibrosis  single-walled carbon nanotubes 
學科別分類
中文摘要 我們先前的研究發現暴露單層奈米碳管會誘發肺部纖維化,在此過程中上皮細胞轉型成間質細胞 (epithelial-mesenchymal transition, EMT) 造成纖維母細胞的增生及肺纖維化的發生。此外,循環成纖維細胞為會表達轉化生長因子(TGF-)及膠原蛋白I (collagenI) 的間質前驅細胞, 可能營造有利於EMT發展之環境。因此,本研究目的是探討循環纖維細胞於單層奈米碳管誘發肺纖維及對EMT發展的貢獻。C57BL6 母鼠以口咽吸入方式暴露單層奈米碳管(80 μg/mouse)至多六週後犧牲,量測肺組織中特定趨化激素及細胞激素,並以流式細胞儀分析不同表型的循環纖維細胞之貢獻。利用免一組隻螢光染色觀察循環纖維細胞分佈,接著以 serum amyloid p (SAP) 抑制循環纖維母細胞之分化以探討循環纖維細胞於此纖維化及EMT形成所扮演之角色。結果顯示肺部中CCL2、CCL12、CXCL12、G-CSF 及 GM-CSF 皆顯著性於暴露一天後上升, 而 CCL21 則是在第三天並分別於4-5週達最高值。因此我們發現表現CCR2+ 及CXCR4+ 表型的循環纖維細胞首先快速的轉移至肺組織中, 接著是以CXCR4+ 及 CCR7+ 表型為主要的循環纖維細胞。於暴露5週後循環纖維細胞達最高峰,並占總纖維母細胞的四分之一。處理SAP會降低循環纖維母細胞於肺部的纖維化、早期施予 SAP 降低 TGF-表現量及EMT比例。並且在免疫螢光染色中觀察到隨著暴露時間增加,循環纖維細胞分化成肌纖維母細胞比例隨之增加。值得注意的是,循環纖維細胞的數目與TGF-的表現量具有顯著相關性。本研究首次證明循環纖維細胞可於體內轉成肌纖維母細胞參與單層奈米碳管誘發肺纖維化過程。
英文摘要 Our previous study found that exposure to single-walled carbon nanotube (SWCNT) causes epithelial-derived fibroblasts account for 42% of total fibroblasts . Moreover, fibrocytes are involved in wound repaire and the development of fibrosis. In this study, we aimed to investigate the contribution of fibrocytes to the development of pulmonary fibrosis during SWCNT-induced fibrogenesis. Female C57BL6 mice were oropharyngeally aspirated with 80 μg SWCNT for up to 6 wk. We characterized the kinetic production of chemokines and cytokines, and phenotyped fibrocytes and their precursor by flow cytometry analysis. Serum amyloid P (SAP) was used to examine the role of fibrocytes in pulmonary fibrosis and EMT. Immunofluorescent stainings were performed for CD45, p-Smda2, JNK, -SMA and Col. Our results demonstrated that following SWCNT exposure, pulmonary CCL2, CCL12, CXCL12, and G-CSF and GM-CSF were significantly increased starting at 1 d, while CCL21 at 3 d. We thus observed the immediate recruitment of CCR2+ and CXCR4+ fibrocytes, with CXCR4+ and CCR7+ fibrocytes becoming predominant later. Fibrocytes accounted for approximately one-fourth of the FSP-1+ fibroblasts at 5 wk. Treatment with SAP attenuated SWCNT-induced pulmonary fibrosis, TGF-β production and EMT. Immunofluorescent staining demonstrated that fibrocytes located to the area of the granulomatous/fibroblastic loci, we for the first time showed that fibrocytes were capable of differentiating into myofibroblasts in vivo. Moreover, pulmonary fibrocyte numbers correlated significantly with the early production of TGF-β, and the development of EMT. In conclusion, fibrocytes contribute to myofibroblast expansion and the occurrence of early EMT during SWCNT-induced pulmonary fibrosis.
論文目次 目錄
頁碼
中文摘要i
英文摘要ii
誌謝.iii目錄.iv
圖目錄vii
附錄目錄.viii
研究內容
第一章 序論
1.1 前言.1
1.2 研究目的.3
第二章 文獻探討
2.1 奈米微粒
2.1.1 奈米科技產業與毒理研究4
2.2 興新奈米微粒與健康效應
2.2.1 單層奈米碳管的發現6
2.2.2 單層奈米碳管的幾何結構與特性6
2.2.3 單層奈米碳管之健康效應與毒性研究7
2.2.4 單層奈米碳管誘發肺部損傷9
2.2.5 肺部纖維化病理機制.10
2.2.6 纖維母細胞的來源及擴張.10
2.2.7 循環纖維細胞.12
第三章 研究材料與方法
3.1 實驗設計架構14
3.2 實驗材料16
3.3 實驗方法16
第四章 結果
4.1 小鼠暴露單層奈米碳管時序性對肺部趨化激素及細胞激素的影響27
4.1.1 小鼠肺組織均質液中 CCL2、CCL12、CXCL12 之表現量
4.1.2 小鼠肺組織均質液中 GM-CSF 和 G-CSF 表現量
4.1.3 小鼠肺組織均質液中纖維轉化因子 (TGF-β) 表現量
4.2 單層奈米碳管的暴露造成循環纖維細胞及其前趨細胞於肺組織和血液中浸潤及累積 28
4.2.1不同表型循環纖維細胞 (fibrocytes) 於周邊血液中累積之情形
4.2.2不同表型循環纖維細胞 (fibrocytes) 於肺組織中累積之情形
4.3 循環纖維細胞及其前驅細胞於肺部和血液中浸潤及累積時序性結果29
4.4 觀察小鼠肺部組織中單層奈米碳管纖維之分布29
4.5 單層奈米碳管暴露後循環纖維細胞於肺部組織中浸潤情形 .29
4.6 循環纖維細胞的浸潤於單層奈米碳管誘發纖維母細胞擴張及肺部纖維化之貢獻.30
4.6.1注射 SAP 抑制劑於單層奈米碳管暴露之小鼠對肺部及血液循環纖維細胞分化之影響
4.6.2觀察單層奈米碳管暴露之小鼠注射 SAP 抑制劑對肺部纖維化之影響
4.6.3 循環纖維細胞於小鼠暴露單層奈米碳管所誘發肺部總纖維母細胞擴張之貢獻比例
4.7小鼠暴露單層奈米碳管肺部組織中循環纖維細胞分化轉型為肌纖維母細胞之比例及 -SMA 表現情形31
4.8 小鼠暴露單層奈米碳管後肺部循環纖維細胞中 p-Smad2及 JNK 之表現情形.32
4.9 循環纖維細胞對EMT發展的影響.32
4.9.1 注射 SAP 抑制劑於單層奈米碳管暴露之小鼠對 EMT 之影響
4.9.2 注射 SAP 抑制劑於單層奈米碳管暴露之小鼠對肺部 TGF-β 表現量之影響
第五章 討論34
第六章 結論40
第七章 參考文獻41
圖表48
附錄65

參考文獻 第七章、參考文獻
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