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系統識別號 U0026-0702201412341900
論文名稱(中文) 藉由樹突細胞來探討六價鉻合併奈米微粒對免疫反應及過敏性接觸性皮膚炎之影響
論文名稱(英文) Immune response and allergic contact dermatitis of chromium (VI) with nanoparticle on dendritic cells activation and function
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 102
學期 1
出版年 103
研究生(中文) 洪時儀
研究生(英文) Shih-Yi Hung
學號 s76004094
學位類別 碩士
語文別 中文
論文頁數 58頁
口試委員 指導教授-王應然
口試委員-王伯智
口試委員-蔡瑞真
口試委員-朱清良
中文關鍵字 過敏性接觸性皮膚炎  樹突細胞  六價鉻  奈米微粒  免疫反應 
英文關鍵字 Allergic contact dermatitis  dendritic cells  chromium(VI)  nanoparticle  immune response 
學科別分類
中文摘要 過敏性接觸性皮膚炎是常見的職業病,其中以六價鉻金屬所導致的為最受注意,這些小分子量的金屬衍生物可被視為一種半抗原而具有致敏的潛力,對於六價鉻的職業暴露,主要途徑為空氣和皮膚的吸收,而另一方面隨著奈米材料應用多元在職業上暴露機會逐漸增加,其途徑也多是透過空氣和皮膚的接觸,因此本實驗目的在於利用樹突細胞的特殊成熟活化性質作為研究平台,以及建立過敏性接觸性皮膚炎動物模式,判定六價鉻所造成的過敏性接觸性皮膚炎是否因為合併奈米微粒而更加嚴重。細胞試驗方面,以MTS assay測量細胞的存活率,並以流式細胞儀分析由小鼠骨髓細胞成功分化為樹突細胞的比例,以及分析樹突細胞的成熟度,另外使用ELISA與RT-PCR測定相關細胞激素的表現,並利用免疫螢光染色觀察細胞自體吞噬現象,以西方墨點法分析相關蛋白的表現,另外也利用RT-PCR觀察TLRs的基因表現。動物試驗方面,建立過敏性接觸性皮膚炎動物模式,量測老鼠耳朵腫脹厚度,再利用組織切片染色觀察皮膚病理變化,以及免疫組織染色觀察細胞激素的變化,並利用局部淋巴結試驗觀察細胞增生情況以及樹突細胞遷移到淋巴結的比例。結果顯示,由小鼠骨髓細胞衍生分化為樹突細胞的比例約為70 %,接著給予樹突細胞不同劑量的六價鉻、奈米二氧化鈦和兩者合併的組別,24小時後觀察存活率,並配合成熟度的結果,最後選擇Cr+6 (150 nM)與TiO2–NP (25 μg/ml)的劑量作為實驗條件,而合併組的細胞激素TNF-α和IL-1β的表現量比起控制組有增加趨勢。由免疫螢光染色觀察合併組LC3的表現有增加,以及利用西方墨點法分析蛋白質的表現,也發現合併組的p62、LC3、Beclin-1、Atg5與TRAF6表現皆上升,利用RT-PCR也觀察到合併組的TLR4與TLR9的基因表現增加,最後利用3-MA將合併組的自體吞噬抑制,觀察細胞激素受到影響。動物實驗方面,合併組的老鼠耳朵厚度顯著大於控制組,由組織切片染色觀察得到合併組表皮層增厚以及細胞浸潤現象,並且有細胞激素釋出的情形。另外觀察到合併組的局部淋巴結重量增加,以及有細胞增生情況,同時偵測局部淋巴結內的樹突細胞比例也最高。根據目前結果顯示,六價鉻合併奈米二氧化鈦具有誘導樹突細胞活化成熟能力,並可能藉由活化TLR4與TLR9的訊息路徑,促使自體吞噬以及細胞激素產生,並進一步將抗原呈現活化T細胞,使過敏性接觸性皮膚炎症狀更為嚴重。
英文摘要 Hexavalent chromium (Cr+6) is widely used in many industries but can induce contact dermatitis in specific occupational conditions especially in cement industries. Besides, application of nanomaterials is increasing recently. Titanium dioxide nanoparticles (TiO2–NP) are some of the most abundantly produced nanomaterials and are found in diverse everyday. The main route of occupational exposure for Cr+6 and TiO2-NP is through the air and skin. The purpose of this study is to determine whether Cr+6 combined with nanoparticles enhances the effects of allergic contact dermatitis (ACD) by using dendritic cells (DCs) and animal model. In ex vivo study, cytotoxicity was analyzed by MTS assay, maturation were determined by flow cytometry, TNF-α was measured by ELISA, autophagy was measured by immunofluorescence, the expression of proteins were performed by western blotting and genes expression were measured by RT-PCR. In in vivo study, animal model for ACD had been established and T cell proliferation and DCs migration were analyzed by local lymph node assay. TNF-α and IL-1β expression in combination group were higher than in control. LC3 was increased in combined treatment group by immunofluorescence. The proteins of p62, LC3, Beclin-1, Atg5 and TRAF6 were increased in combination group. TLR4 and TLR9 mRNA levels were increased in combination group. ACD symptoms were found more significant in the combination group. These results show that Cr+6 combined with TiO2-NP could have the potential to induce more dendritic cells activation and might result in severe ACD response by activating TLR4 and TLR9 signaling pathway, inducing autophagy and cytokine production.
論文目次 目錄
第一章、序論 1
第二章、文獻回顧 2
第一節、接觸性皮膚炎(Contact dermatitis)之分類 2
第二節、過敏性接觸性皮膚炎(allergic contact dermatitis)之可能機轉 3
第三節、重金屬六價鉻(Hexavalent chromium, Cr+6) 4
第四節、奈米二氧化鈦(Titanium dioxide nanoparticle, TiO2-NP) 5
第五節、樹突細胞 (Dendritic cell) 6
第六節、T細胞 7
第七節、自體吞噬(Autophagy)與免疫反應 8
第三章、研究目的 11
第四章、研究架構 12
第一節Ex vivo 12
第二節In vivo 13
第五章、研究材料與方法 14
第一節、研究材料 14
第二節、研究方法與實驗步驟 20
一、樹突細胞製備(Generation of DC) 20
二、細胞存活率 (Cell viability) 20
三、樹突細胞分化成功比例與成熟度(Maturation)分析 21
四、細胞激素(Cytokine)分析 21
五、免疫螢光染色(Immunofluorescence) 22
六、西方墨點法(Western blotting) 22
七、反轉錄聚合酶鏈反應(RT-PCR) 24
八、六價鉻誘導過敏性接觸性皮膚炎之動物模式 25
九、蘇木紫伊紅染色(Hematoxylin & Eosin Staining) 26
十、免疫組織染色(Immunohistochemistry) 27
十一、局部淋巴結試驗(Local lymph node assay) 27
十二、樹突細胞遷移(Migration)比例分析 28
十三、統計分析 29
第六章、研究結果 30
第一節、樹突細胞分化培養 30
第二節、樹突細胞暴露六價鉻、奈米二氧化鈦與兩者合併之存活率 30
第三節、樹突細胞暴露六價鉻、奈米二氧化鈦與兩者合併之成熟度變化 31
第四節、樹突細胞暴露六價鉻、奈米二氧化鈦與兩者合併之細胞激素(cytokines)變化情況 32
第五節、六價鉻、奈米二氧化鈦與兩者合併活化樹突細胞TLRs訊息傳遞路徑 32
第六節、利用免疫螢光染色觀察各組暴露物對樹突細胞自體吞噬作用現象 33
第七節、六價鉻、奈米二氧化鈦與兩者合併對樹突細胞之自體吞噬相關蛋白的表現以及對細胞激素的影響 33
第八節、過敏性接觸性皮膚炎之動物模式 34
第九節、觀察過敏性接觸性皮膚炎動物模式的局部淋巴結細胞增生現象以及樹突細胞遷移現象 35
第七章、討論 37
第八章、結論與建議 40
第九章、參考文獻 41
圖表 46



圖表目錄
圖 一、過敏性接觸性皮膚炎致敏階段與誘發階段示意圖(Patel et al. 2012) 3
圖 二、樹突細胞受刺激後活化成熟示意圖 7
圖 三、樹突細胞與T細胞前後關係示意圖(Kaplan et al. 2012) 8
圖 四、自體吞噬與免疫反應(Hisamatsu et al. 2013) 9
圖 五、TLR訊息傳遞路徑圖(Shi and Kehrl 2010) 10
圖 六、細胞內自噬體與MHCⅡ融合示意圖(Levine and Deretic 2007) 10

Figure 1. Mouse bone marrow-derived DCs were observed on day 7 by microscope. 46
Figure 2. DCs viability were measured by MTS assay. 47
Figure 3. Measurement of DCs maturation. 48
Figure 4. Measurement of the cytokines expression. 51
Figure 5. Analysis of TLR4 and TLR9 signaling pathway in DCs. 52
Figure 6. Measurement of autophagy in DCs by immunofluorescence for LC3. 53
Figure 7. Analysis of autophagic-related proteins expression in DCs by western blot and the cytokines mRNA levels by RT-PCR. 54
Figure 8. Results of the elicitation after 96 hrs in animal model. 55
Figure 9. The expression of TNF-α were determined by immunohistochemistry. 56
Figure 10. The migration of DCs to local LNs was increased in chromium alone and combined treatment. 57



Table 1. Analysis of T cell proliferation at the local lymph nodes in chromium alone and combined treatment . 58
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