近年來空氣汙染議題備受矚目，其中雲林地區空汙嚴重程度佔全國的前五名，而產生空氣汙染的原因主要為本地、境外的汙染物、氣候不良及地形條件等成因。本研究欲探討雲林地區的細懸浮微粒(PM2.5)，對於人體支氣管肺上皮細胞(BEAS-2B)的危害，以及慢性阻塞性肺病(COPD)的風險評估，並且透過巴西蘑菇萃取液與PM2.5作用，觀察是否具有改善的PM2.5危害的效果。首先進行雲林虎尾地區的細懸浮微粒(PM2.5)採集及濃度計算，接著將巴西蘑菇依不同萃取方式(熱水萃取、酵素萃取、超高壓輔助酵素萃取)進行提取，探討不同萃取方式的多醣含量以及抗氧化能力，藉由總醣與還原醣測定進行多醣含量的估算，實驗結果發現超高壓輔助酵素萃取的多醣產量高達34.11%。在抗氧化能力試驗中，透過清除DPPH自由基、螯合亞鐵離子試驗，評估樣品的供氫能力與減少脂質過氧化能力，實驗結果發現超高壓輔助酵素萃取具有較好的抗氧化能力，在清除DPPH自由基試驗中樣品濃度1000 μg/ml，抑制率達到84.61%，清除效果稍微高於正控制組的表現。將樣品與採集的PM2.5進行細胞實驗，透過暴露於BEAS-2B細胞進行細胞毒性、發炎因子(IL-6、IL8)及COPD試驗，由MTT試驗得知，巴西蘑菇超高壓輔助酵素萃取液在濃度為0.2 mg/ml時對於BEAS-2B細胞不具細胞毒性，而PM2.5於濃度15.04 μg/ml時產生細胞毒性，將0.2 mg/ml巴西蘑菇萃取液與PM2.5 (15.04 μg/ml)共同暴露於BEAS-2B細胞培養24hr，能使存活率提高至96%，由此可推斷巴西蘑菇具有保護BEAS-2B的趨勢。而在氧化壓力試驗中，PM2.5產生的氧化壓力是控制組的11倍，但加入巴西蘑菇萃取液後，能減少約10%的ROS產生。接著進行發炎因子IL-6、IL-8評估，實驗結果發現PM2.5對於IL-6、IL-8的表達顯著，表示PM2.5會使BEAS-2B產生發炎反應，當加入巴西蘑菇萃取液共同培養時，PM2.5產生的發炎因子具有顯著下降的趨勢。並且針對COPD相關標的物α-1-抗胰蛋白酶(AAT)進行表達測定，發現PM2.5會使AAT的表達下降，但是加入巴西蘑菇萃取液能使AAT的表達具有顯著上升，推測能降低COPD風險的趨勢，綜合上述結果發現，巴西蘑菇對於PM2.5在肺部所產生的細胞毒性、氧化壓力、抗發炎反應及COPD的危害能達到稍微改善的效果，具有開發相關保健食品的潛力。

The air pollution issue has attracted much attention in recent years. Among other county, the severity of air pollution in Yunlin area accounts for the top five in Taiwan country. The main reason of the air pollution is Local and foreign pollutants, poor climate, and topographic. In this experiment, we were investigate the particulate matter (PM2.5) in the Yunlin area, the risk to human bronchopulmonary epithelial cells (BEAS-2B), and the assessment of chronic obstructive pulmonary disease (COPD). And through the action of Agaricus blazei Murill extract and PM2.5, observe whether there is an effect of improved PM2.5 hazard. Firstly, the particulate matter (PM2.5) collection and concentration calculation in the Huwei area of Yunlin are carried out. Then, the Agaricus blazei Murill are extracted by different extraction methods (hot water extraction, enzyme extraction, ultra-high pressure auxiliary enzyme extraction). To explore the polysaccharide content and antioxidant capacity of different extraction methods, and to estimate the polysaccharide content by total sugar and reducing sugar. The experimental results showed that the yield of polysaccharide extracted by ultra- high pressure auxiliary enzyme was as high as 34.11%. In the antioxidant capacity test, we evaluated the hydrogen supply capacity of the sample and reduced the lipid peroxidation ability by removing DPPH free radicals and chelated ferrous ions. The experimental results showed that the ultra-high pressure auxiliary enzyme extraction had better antioxidant capacity. The sample concentration was 1000 μg/ml in the DPPH free radical scavenging test, and the inhibition rate reached 84.61%. The scavenging effect was slightly higher than that of the positive control group. The sample was subjected to cell experiments with the collected PM2.5, and the cytotoxicity, inflammatory factor (IL-6, IL8) and COPD test were carried out by exposure to BEAS-2B cells. The MTT test showed that the Agaricus blazei Murill ultra-high pressure auxiliary enzyme extract. It is not cytotoxic to BEAS-2B cells at a concentration of 0.2 mg/ml, while PM2.5 produces cytotoxicity at a concentration of 15.04 μg/ml. 0.2 mg/ml Brazilian mushroom extract with PM2.5 (15.04 μg/ml). In the oxidative stress test, PM2.5 produced 11 times the oxidation pressure of the control group, but the addition of the Brazilian mushroom extract reduced the ROS production by about 10%. Then the inflammatory factors IL-6 and IL-8 were evaluated. The results showed that PM2.5 was significantly expressed in IL-6 and IL-8, indicating that PM2.5 produced an inflammatory reaction in BEAS-2B when adding Brazilian mushroom extract. When co-cultured, the inflammatory factors produced by PM2.5 showed a significant downward trend. Furthermore, the expression of AAT was detected by PM2.5, and it was found that PM2.5 decreased the expression of AAT, but the addition of Brazilian mushroom extract could significantly increase the expression of AAT, which was supposed to reduce COPD. The trend of risk, combined with the above results, found that Brazilian mushrooms have a slightly improved effect on the cytotoxicity, oxidative stress, anti-inflammatory response and COPD caused by PM2.5 in the lungs, and have the potential to develop relevant health foods.

摘要.....................................................i
Abstract................................................iii
誌謝.....................................................v
目錄.....................................................vi
表目錄...................................................ix
圖目錄...................................................x
縮寫對照表...............................................xi
第一章 緒論..............................................1
1-1研究動機..............................................1
1-2研究目的...............................................1
第二章 文獻回顧...........................................2
2-1懸浮微粒(PM)...........................................2
2-1-1懸浮微粒介紹..........................................2
2-1-2 PM2.5之來源與組成....................................2
2-1-3雲林地區PM2.5空氣品質.................................2
2-2懸浮微粒(PM)對於肺部的相關危害...........................3
2-2-1 懸浮微粒對肺部之侵入方式與危害........................3
2-2-2氧化壓力.............................................3
2-2-3慢性阻塞性肺病(COPD)..................................3
2-2-4 α-1-抗胰蛋白酶(AAT)..................................4
2-2-5發炎因子介紹..........................................4
2-3真菌多醣體..............................................5
2-3-1真菌多醣介紹..........................................5
2-3-2真菌多醣萃取方法......................................5
2-4高壓萃取...............................................6
2-4-1高壓加工技術..........................................6
2-4-2超高壓輔助酵素萃取.....................................6
2-5巴西蘑菇................................................6
2-5-1巴西蘑菇介紹..........................................6
2-5-2巴西蘑菇之功效........................................7
2-6自由基對於生理作用之影響.................................8
2-6-1自由基之介紹..........................................8
2-6-2自由基之種類..........................................8
2-6-3自由基之來源..........................................8
2-6-4自由基對生物體之影響...................................9
2-7抗氧化劑作用............................................9
2-7-1抗氧化劑應用..........................................9
2-7-2抗氧化劑作用原理......................................9
1.自由基終止劑.............................................9
2.還原劑或氧清除劑.........................................10
3.金屬螯合劑..............................................10
4.單重態氧的抑制劑.........................................10
第三章 實驗設計............................................11
3-1 實驗規劃..............................................11
3-2 實驗架構..............................................12
第四章 材料與方法..........................................13
4-1 實驗材料..............................................13
4-1-1 巴西蘑菇(Agaricus blazei Murill)粉末................13
4-1-2 酵素...............................................13
4-1-3 實驗細胞...........................................13
4-1-4 藥物試劑...........................................13
4-2 實驗方法.............................................14
4-2-1 PM2.5顆粒採樣.......................................14
4-2-2 PM2.5顆粒萃取......................................14
4-2-3巴西蘑菇萃取液製備....................................14
1.巴西蘑菇前處理..........................................14
2.巴西蘑菇熱水萃取物製備...................................14
3.巴西蘑菇酵素萃取物製備...................................14
4.巴西蘑菇高壓輔助酵素萃取物製備............................15
4-2-4 總醣含量測定(Phenol-sulfuric acid)..................15
4-2-5 還原醣含量測定(DNS method)..........................15
4-2-6 多醣含量換算........................................16
4-2-7 抗氧化能力測定......................................16
1.清除DPPH自由基能力之測定................................16
2.螯合亞鐵離子能力之測定...................................16
4-2-8 細胞實驗...........................................17
1.細胞培養...............................................17
2.細胞存活率測試(MTT試驗)..................................17
3.細胞活性氧化物測定(ROS試驗)..............................17
4-2-9酵素結合免疫吸附分析法(ELISA Assay)...................18
1. 發炎因子IL-6測定.......................................18
2. 發炎因子IL-8測定.......................................18
3. α-1-antitrypsin (AAT) 測定............................19
4-2-10統計分析...........................................19
第五章 結果與討論.........................................20
5-1巴西蘑菇酵素萃取液單因子評估(時間、溫度、用量)............20
5-1-1比較酵素於不同時間水解之多醣含量......................20
5-1-2比較酵素於不同溫度水解之多醣含量......................20
5-1-3比較酵素不同用量水解之多醣含量........................20
5-2不同萃取方式巴西蘑菇萃取液醣量比較......................21
5-3 抗氧化能力評估.......................................22
5-3-1清除DPPH自由基能力評估...............................22
5-3-2螯合亞鐵離子能力評估.................................22
5-4 顆粒採樣............................................23
5-5 細胞毒性測定........................................23
5-5-1巴西蘑菇萃取液對BEAS-2B細胞毒性(MTT assay)...........23
5-5-2 PM2.5對BEAS-2B細胞毒性(MTT assay).................24
5-5-3 巴西蘑菇與PM2.5共同培養對BEAS-2B細胞毒性............24
5-5-4 巴西蘑菇與PM2.5共同培養減少對BEAS-2B細胞ROS生成量...25
5-5-5 巴西蘑菇與PM2.5共同培養對BEAS-2B細胞的發炎反應評估...26
1. 發炎因子IL-8測定.....................................26
2. 發炎因子IL-6測定.....................................26
5-5-6 巴西蘑菇與PM2.5共同培養對BEAS-2B細胞的AAT蛋白表現....26
第六章 結論.............................................28
參考文獻................................................29
表附錄 ...............................................36
圖附錄 ...............................................45
Extended Abstract......................................68

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