臺灣紫芝 (Ganoderma formosanum) 是臺灣特有種靈芝，其萃取物具有多種生理活性。本研究通過液態發酵的方式來大量生產臺灣紫芝菌絲體，以95%酒精對其菌絲體進行萃取，並利用人類皮膚角質細胞 (HaCaT cell) 探討經液態發酵之菌絲體萃取物 (G. formosanum mycelial extract, GFE) 對PM2.5 (fine particulate matter) 所引起之活性含氧物生成以及細胞傷害之調控功能，並進一步利用斑馬魚動物模型證實PM2.5對於動物體的傷害。傷害。
研究結果顯示500 μg/mLppm的PM2.5會導致胞內ROS (reactive oxygen species) 增加2.7倍且導致細胞存活率下降45%，且具有dose-dependent效應；而在斑馬魚體內也可以觀察到相似的效應，2,000 μg/mLppm的PM2.5會導致斑馬魚體內的ROS增加1.7倍，且會引起斑馬魚心率下降；加入臺灣紫芝萃取物處理後，PM2.5所引起的胞內ROS的水平顯著減少59%，同時減少30%的細胞凋亡；在斑馬魚動物模型中，經臺灣紫芝萃取物處理後斑馬魚體內之ROS水平降低了67%。以流式細胞儀分析也顯示臺灣紫芝萃取物之預處理可以有效減少細胞凋亡。綜上所述，臺灣紫芝萃取物可透過抗氧化活性從而保護皮膚細胞不受PM2.5損傷。

Ganoderma formosanum is an endemic species of Ganoderma in Taiwan and extract of G. formosanum possesses diverse bioactivities. This study discussed about the potential protective effect of submerged cultivated G. formosanum mycelial extract (GFE) against PM2.5-induced ROS generation and cell viability in human keratinocyte (HaCaT cell) model, meanwhile assessed the toxicity of PM2.5 on zebrafish in vivo model.
Results showed that PM2.5 treatment promoted both the intracellular ROS level and decreased cell viability in a dose-dependent manner. The similar effect could also be observed in zebrafish model. Pretreatment of G. formosanum extract ameliorated intracellular ROS generation and apoptosis. The PM2.5-induced cell damage was attenuated by antioxidant N-acetyl cysteine, confirming that PM2.5 cellular toxicity was due to oxidative stress. Taken together, these findings implied that antioxidant activities of G. formosanum extract might play an important role in the protection of PM2.5-induced cell damage.

目錄
謝誌 I
摘要 III
Abstract IV
目錄 VI
圖目錄 IX
表目錄 XI
List of Figures XII
List of Tables XIV
壹、前言 1
貳、文獻回顧 3
一、靈芝 3
1.1 靈芝的種類 3
1.2 靈芝的生理活性 5
1.3 靈芝的活性成分 5
二、臺灣紫芝 9
三、靈芝培養方式 12
3.1 靈芝固態培養 12
3.2 靈芝菌液態培養 13
四、空氣污染 13
4.1 粒狀物質 (particulate matter, PM) 15
4.2 粒狀物質的組成 18
4.3 對粒狀物質的限制 18
4.4 粒狀物質對皮膚之傷害 19
4.5 粒狀物質對細胞的影響 21
五、斑馬魚動物模型 23
六、活性含氧物 (Reactive oxygen species) 25
七、研究目的 27
參、實驗架構 29
肆、材料與方法 32
一、臺灣紫芝液態發酵生產 32
1.1 實驗藥品 32
1.2 儀器設備 33
1.3 實驗菌株 34
1.4 培養基 34
1.5 培養方法 35
1.6 各活性劃分之分離 36
二、臺灣紫芝各活性劃分用於曝露於粒狀物質之皮膚細胞存活率測定 37
2.1 實驗藥品 37
2.2 儀器設備 38
2.3 實驗細胞株 38
2.4 細胞培養基 39
2.5 細胞培養 40
2.6 細胞存活率之測定 42
2.7 細胞胞內ROS之測定 42
2.8 PM2.5的製備 43
三、萃取物各活性劃分之活性探討 43
3.1各活性劃分用於曝露於PM2.5之皮膚細胞存活率測定 43
3.2各活性劃分用於曝露於PM2.5之皮膚細胞胞內ROS之測定 45
3.3 流式細胞儀分析細胞凋亡相關表現 45
四、斑馬魚培養 48
4.1 培養方法 48
4.2 斑馬魚體內ROS測定 48
五、統計分析 49
伍、結果與討論 50
一、PM2.5之形態 50
二、PM2.5之毒性 50
2.1對細胞存活率之影響 50
2.2對胞內ROS之影響 53
2.3 對斑馬魚之影響 55
2.4 PM2.5所引起之氧化壓力 56
三、臺灣紫芝萃取物抗PM2.5之活性探討 59
3.1臺灣紫芝萃取物抗PM2.5活性之初篩 59
3.2臺灣紫芝萃取物之毒性探討 59
3.3 臺灣紫芝萃取物各活性劃分抗PM2.5之活性探討 62
四、流式細胞儀分析細胞凋亡 67
4.1 PM2.5所造成的傷害 67
4.2 臺灣紫芝萃取物的保護效果 67
陸、結論與未來展望 72
柒、參考文獻 73
捌、附錄 XV
附錄一 XV
履歷表 XVIII
附錄二 XIX

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