題目一、 建立進階評估客雅溪底泥污染物毒性的細胞平臺
美國環境保護署（United States Environmental Protection Agency, USEPA） 於2000 年的報告中，將底泥列為河流、溪流、湖泊和水庫中主要的污染物之一。底泥
容易被工業廢水、生活污水以及農業廢水所影響，一旦這些污染源進入水體之中，
就容易被底泥吸附並且很難藉由自然降解而脫附。
研究中，我們對客雅溪的上游、中游和下游，三個採樣點進行分析與評估，並了解其對於人體細胞的影響。我們總共選用了 11 種不同的細胞，在此只顯示我個人主要研究的肺部細胞 – A549 和 BEAS-2B 的實驗成果。結果顯示，客雅溪下游的底泥污染比其他兩者嚴重。處理過後的下游底泥萃取物，使 A549 和 BEAS-2B 的細胞活力皆顯著降低；此外，貼附能力亦是明顯地下降，其中以 BEAS-2B 為最；而傷口癒合能力的部分，除了 BEAS-2B 因為其細胞特性的原因不適合此實驗外，A549 的爬行能力也是明顯地降低許多。我們的結果顯示，客雅溪下游的底泥無論是物理特性還是化學分析上皆與上游和中游有較大的差異，對於細胞的影響亦是較其他兩者嚴重。兩者合計，我們希望建立一個人類細胞的平臺，用以評估底泥的生物毒性，進一步了解生態環境對人類健康的影響。

題目二、 Raw 264.7 巨噬細胞對於懸浮微粒的生物效應
人類開發環境創造舒適文明生活的同時，自然環境也因為人類活動所造成的大量污染，導致生態體系被破壞，甚至是反撲至人類身上，危及人體健康。直至 1950年代，人們才開始注意到懸浮微粒所造成的危害。雖然許多研究已證實懸浮微粒（particulate matter, PM ）會對呼吸系統和心血管系統造成傷害，但截至目前為止針對懸浮微粒之於免疫系統的相關研究卻非常稀少，還有很多機制尚未釐清。
本研究中選用美國國家標準技術研究院（National Institute of Standards and
Technology, NIST）的標準參考物質（Standard reference materials, SRMs） - SRM 2786 ( PM 4 ) 、 SRM 2787 ( PM 10 ) 以及小鼠的巨噬細胞 – RAW 264.7 作為材料，來探討在暴露於不同濃度、不同時間、不同頻率的懸浮微粒條件下，對於細胞存活率的影響和 RAW 264.7 細胞的吞噬現象、趨化性及細胞激素分泌之間的關聯性。本研究的結果表明，隨著懸浮微粒暴露濃度的增加，對於 RAW 264.7 細胞的細胞存活率、趨化能力以及細胞激素分泌量的影響亦跟著增加。然而有趣的是，在細胞存活率的部分，PM 4 對細胞存活率的影響大於 PM 10；但是在趨化性實驗及細胞激素分泌測定的部分，PM 10 對於細胞的影響反而是大於 PM 4。本研究欲藉由嚴謹的小鼠免疫體外測定模式，協助人們瞭解懸浮微粒對生物體健康的影響，以準確地評估懸浮微
粒對人體的毒性，並提供環境預防醫學上政策制定的科學依據。

Project 1. Establishment of cell-based platforms for advancing effectiveness evaluation in toxicity assessment of Ke-Ya Creek sediment pollutants.
While human over-consume environment to enable a wealthier and more comfortable civilized life, they also cause a lot of damage to pollute their natural ecosystems and hazard human health. Nevertheless, most of the current research and bioavailability are based on the changing numbers in distribution and the community
composition of plankton, benthos and fish.
For direct investigation of sediment toxicity, this study takes advantage of human cell lines which are commonly used in the implementation of human health assessment in pharmacological and toxicological studies. We evaluated the effects of three sediment
sampling points upstream, midstream, and downstream of Ke-Ya Creek on different cell lines. There were 11 different cell lines used in this project. Herein, lung cell lines, A549 and BEAS-2B, were demonstrated. Accordingly, the cell viability and the adhesion ability of A549 and BEAS-2B was significantly reduced after the treatment of downstream
sediment extracts compared to other locations. Furthermore, the wound healing ability of A549 was also significantly reduced due to the presence of 1 % downstream organic sediment extract.
Apparently, our results indicated that the sediment pollution of Ke-Ya downstream was more toxic than that of the upstream and midstream. Taken together, we have established a cell-based platform to assess the biotoxicity of the sediment for further the impact of river sediment pollution on human health risk as a basis for further evaluation of ecological environment.

Project 2. Biological effects of particulate matter in Raw 264.7 macrophages
While human beings develop the environment and create a comfortable and civilized life, they also lead to the destruction of ecological systems and endanger human health. Although many studies have confirmed that particulate matter (PM ) can cause damage to
the respiratory and cardiovascular systems, research on the immune system has so far been scarce, and many molecular mechanisms have not yet been clarified.
In this study, National Institute of Standards and Technology's standard reference materials SRM 2786 (PM 4) and SRM 2787 (PM 10) are utilized as the airborne suspended particles to investigate the reactions to immune cell Raw 264.7 macrophages. The composition of heavy metals and organic pollutants in PM , as well as cytotoxicity
and cytokines of treated immune cells are analyzed. Our results indicated that when the exposure concentration of PM increases, the effect on the cell viability, chemotaxis and cytokine secretion of RAW 264.7 cells are also increased. Furthermore, PM 4 has a greater impact on cell survival than PM 10; but in the chemotaxis and cytokine assay, PM 10 has a stronger effect instead of PM 4.
Through the rigorous mouse immune cell lines , we hope to clearly evaluate the effects of suspended particles of different concentrations and particle sizes on the immune functions at different stages of stimulation, thereby assessing the harm of PM to human
immune system and providing the scientific basis to adopt strategies for disease prevention and policies making for environmental protection.

題目一、 建立進階評估客雅溪底泥污染物毒性的細胞平臺
目錄
圖目錄..... VI
表目錄..... VII
1. 前言..... 1
2. 材料和方法..... 3
2.1 樣品採集地點..... 3
2.2 底泥物理性質分析..... 4
2.2.1 pH值..... 4
2.2.2 含水量..... 4
2.2.2 質地分析..... 4
2.3　底泥化學性質分析..... 5
2.3.1 有機碳含量..... 5
2.3.2 陽離子交換容量..... 5
2.4 底泥污染物分析..... 5
2.5 底泥萃取..... 6
2.6 細胞介紹和細胞培養..... 7
2.7 細胞存活率測定..... 8
2.8 細胞貼附試驗..... 9
2.9 傷口癒合實驗..... 9
2.10 統計分析..... 10
3. 結果..... 11
3.1底泥的物化性質..... 11
3.2 底泥萃取液的毒性與細胞存活率..... 14
3.3 底泥萃取液對細胞貼附能力的影響..... 17
3.4 底泥萃取液對細胞傷口癒合影響..... 18
4. 討論.....21
5. 結論.....24
參考資料..... 25

圖目錄
圖1. 採樣相對位置圖....... 3
圖2. 使用之細胞圖...... 8
圖3. 上中下游的土壤質地圖.......12
圖4. 細胞存活率分析圖.......15
圖5. 細胞貼附測定圖.......17
圖6. A549傷口癒合測定圖.......19
圖7. A549傷口癒合面積統計圖.......19
圖8. BEAS-2B細胞圖.....20

表目錄
表一、上中下游三個採樣點底泥污染物的物化特性....11
表二、底泥重金屬污染物分析....13
表三、底泥有機污染物分析 ....14


題目二、 Raw 264.7 巨噬細胞對於懸浮微粒的生物效應
目錄
圖目錄..... VII
表目錄..... VIII
1. 前言..... 1
2. 材料與方法..... 4
2.1 環境標準參考物質 ......4
2.2 懸浮微粒樣品配置...... 4
2.3 掃描式電子顯微鏡觀察環境標準參考物質...... 4
2.4 小鼠巨噬細胞Raw 264.7之來源與培養...... 5
2.4 細胞存活率測定（MTT cell viability assay)...... 5
2.5 暴露頻率試驗（Exposure frequency assay）...... 6
2.6 螢光顆粒與細胞免疫螢光染色（Immunofluorescence assay）...... 7
2.7 細胞免疫行為測試...... 8
2.7.1 細胞趨化性試驗 ( Chemotaxis assay )...... 9
2.7.2 細胞激素測定 ( Cytokine profiling )...... 9
2.7.2.1 西方墨點法 ( Western Blotting )..... 10
2.7.2.2 Bio-Plex Cytokine Assay.....11
2.8 統計分析..... 11
3. 結果..... 12
3.1 環境標準參考物質之物化性質..... 12
3.2 懸浮微粒的毒性與細胞存活率..... 14
3.3 懸浮微粒的暴露頻率試驗..... 15
3.4 RAW 264.7細胞的免疫螢光染色..... 16
3.5 趨化性試驗..... 18
3.6 細胞激素測定 - 西方墨點法..... 19
3.7 細胞激素測定 - Bio-Plex Cytokine Assay..... 20
4. 討論..... 23
5. 結論..... 25
參考資料 .....27

圖目錄
圖1. SRM的顆粒形狀、大小與粒徑分布圖 ....... 13
圖2. 細胞存活率分析圖 ...... 14
圖3. 暴露頻率試驗細胞存活率分析圖 ...... 16
圖4. RAW 264.7細胞暴露於綠色螢光染色聚苯乙烯顆粒時的免疫螢光染色照片 ..... 17
圖5. RAW 264.7細胞暴露於不同濃度懸浮微粒的趨化性試驗染色照片 ...... 18
圖6. RAW 264.7細胞暴露於不同濃度懸浮微粒的趨化性試驗染色統計圖表 ..... 19
圖7. RAW 264.7細胞之細胞激素測定結果 ( 西方墨點法 ) ...... 20
圖8. RAW 264.7細胞之細胞激素測定結果 ( Bio-Plex Cytokine Assay ) ...... 22
圖9. 巨噬細胞遭遇懸浮微粒後可能引發之免疫反應關係圖..... 26
 
表目錄
表一、暴露頻率實驗設計 ....... 7

題目一、 建立進階評估客雅溪底泥污染物毒性的細胞平臺
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題目二、 Raw 264.7 巨噬細胞對於懸浮微粒的生物效應
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