臺灣博碩士論文加值系統

本實驗將利用肺細胞 in vitro 系統進行 WO3 及 WO3·H2O 之肺毒性評估，並探討其相關生物作用機制。細胞毒性實驗結果顯示兩種材料對肺癌細胞之毒性作用皆有不同程度之劑量效應。兩種材料對肺癌細胞誘發之細胞凋亡皆有不同程度之劑量效應。其中以 WO3 可於肺癌細胞誘發最多的細胞凋亡及最高的細胞毒性。本實驗亦發現兩種材料皆會使肺癌細胞內 HO-1 及 SOD-2 表現明顯下降。細胞內 HO-1 及 SOD-2 若無法正常表達將導致細胞內的 ROS 大量累積。實驗進一步檢測細胞內 DNA 損害和 IL-6 發炎因子的表達量，兩種材料皆誘發顯著的細胞 DNA 損傷以及 IL-6 發炎因子表現。接著是 WO3 及 WO3·H2O 之細胞攝取能力評估，然而只有 WO3 具有顯著的細胞攝取能力。最後實驗檢測細胞內 SERPINA-1 表現量，兩種材料對SERPINA-1 表現量皆有明顯下降，代表 WO3 及 WO3·H2O 具有誘導 COPD 的可能性。
　　綜合上述實驗結果顯示，WO3·H2O 相較於 WO3 有較低的毒性。本實驗證明 WO3 改質後，可以降低其毒性，並顯示出 WO3·H2O 在未來的應用上的發展潛力。

In this experiment, tungsten trioxide (WO3) was modified to synthesize tungsten trioxide hydrate (WO3·H2O). The lung cytotoxicity test of WO3 and WO3·H2O will be evaluated in this study and its mechanism will be explored. Results indicated that WO3 and WO3·H2O induced significant decreases in cell viability (<86% of control) and significant increases in LDH leakage (>110% of control) after 24 hr exposure. Both of the materials reduced the expression of HO-1 (0.15-0.33 folds of control) and SOD-2 (0.31-0.66 folds of control) that increased the production of proinflammatory cytokins (1.6-2.3 folds of control) as well as decrease SERPINA-1 expression (0.28-0.58 fold of control). The results show that WO3·H2O has lower toxicity than WO3. This study proves that the modified WO3 reduce its toxicity and show the development potential of WO3·H2O in future applications.

摘要 ............................................................................................................................................. i
Abstract ....................................................................................................................................... ii
誌謝 ........................................................................................................................................... iii
目錄 ........................................................................................................................................... iv
表目錄 ....................................................................................................................................... vi
圖目錄 ...................................................................................................................................... vii
縮寫對照表 ............................................................................................................................. viii
第一章 緒論 .............................................................................................................................. 1
1.1 研究緣起 ...................................................................................................................... 1
1.2 研究目的 ...................................................................................................................... 2
第二章 文獻回顧 ...................................................................................................................... 3
2.1 奈米材料 ...................................................................................................................... 3
2.2 奈米科技發展潛在危險 .............................................................................................. 7
2.3 三氧化鎢 (Tungsten Trioxide,WO3)介紹 .................................................................. 8
2.4 慢性阻塞肺疾病 (COPD) ........................................................................................... 8
2.5 氧化壓力(Oxidative stress) .......................................................................................... 9
2.6 活性氧 (Reactive Oxygen Species，ROS) .............................................................. 10
第三章 材料與方法 ................................................................................................................ 12
3.1 實驗流程 ................................................................................................................... 12
3.2 實驗藥品 ................................................................................................................... 12
3.3 實驗器材 ................................................................................................................... 13
3.4 合成WO3·H2O .......................................................................................................... 13
3.5.三氧化鎢之相關專利 ................................................................................................ 13
3.6 TEM 樣品製備 ........................................................................................................... 14
v
3.7 動態光散射粒徑分析儀(DLS) ................................................................................. 14
3.8 細胞培養 ................................................................................................................... 14
3.9 細胞存活率試驗(MTT 試驗) ................................................................................... 14
3.10 乳酸脫氫酶試驗(LDH 試驗) ................................................................................. 15
3.11 流式細胞儀 ............................................................................................................. 15
3.12 酵素結合免疫吸附分析法(ELISA Assay) ............................................................. 16
第四章 結果與討論 ................................................................................................................ 17
4.1 WO3 及 WO3·H2O 奈米顆粒物理性質分析 .......................................................... 17
4.1.1 WO3 及 WO3·H2O 奈米顆粒之TEM 結果分析圖 ...................................... 17
4.1.2 WO3 及 WO3·H2O 奈米顆粒之水合粒徑測定 ........................................... 18
4.1.3 WO3 與 WO3·H2O 奈米顆粒之表面電位之分析 ....................................... 19
4.2 WO3 與 WO3·H2O 奈米顆粒於人類肺癌細胞中誘發之細胞毒性測試 .............. 21
4.2.1 WO3 與WO3·H2O 奈米顆粒之MTT 試驗 .................................................. 21
4.2.2 WO3 及WO3·H2O 奈米顆粒之LDH 試驗 .................................................... 23
4.3 WO3 及WO3·H2O 奈米顆粒於人類肺癌細胞中誘發之細胞凋亡 ......................... 24
4.4 WO3 及WO3·H2O 奈米顆粒於人類肺癌細胞中誘導抗發炎及抗氧化因子表現 . 26
4.4.1 WO3 及WO3·H2O 誘導人類肺癌細胞抗發炎及抗氧化因子表現(HO-1) .. 26
4.4.2 WO3 及WO3·H2O 誘導人類肺癌細胞抗發炎及抗氧化因子表現(SOD-2) 27
4.5 WO3 及WO3·H2O 奈米顆粒於人類肺癌細胞中誘發細胞DNA 損傷評估 .......... 28
4.6 WO3 及WO3·H2O 奈米顆粒於人類肺癌細胞中誘發細胞發炎反應 (IL-6) ......... 30
4.7 WO3 及WO3·H2O 奈米顆粒於人類肺癌細胞中之細胞攝取能力評估 ................. 31
4.8 WO3 及 WO3·H2O 奈米顆粒誘發 COPD 相關基因表現 ................................... 32
第五章 結論 ............................................................................................................................ 33
參考文獻 .................................................................................................................................. 34
Extended Abstract ..................................................................................................................... 39

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