Study of ganoderic acid and volatiles biosynthesis during heat stress-induced apoptosis

靈芝是一種已經長時間在亞洲國家所使用珍貴中藥材，其主要藥物成分靈芝酸 (ganoderic acids) 是屬於三萜類化合物，目前被證實具有抗癌、抗發炎和保肝等功效。在植物中發現真菌細胞壁可誘導植物細胞凋亡及二次代謝物產生。然而，真菌中調控細胞凋亡與二次代謝物生合成的相關研究甚少。本研究想了解高溫誘導靈芝細胞凋亡及靈芝酸生合成的可能調控機轉，探討細胞膜流動、鈣離子訊息傳遞所扮演的角色。我們的研究成果證實利用鈣離子螢光染劑 (Fluo-3AM) 證實在高溫下靈芝細胞內鈣離子濃度上升，而50 mM EGTA (ethylene glycol-bis (β-aminoethyl ether) -N,N,N',N'-tetraacetic acid)前處理則可降低高溫所造成細胞內鈣離子濃度的上升；利用HPLC進行代謝物分析，證實50 mM EGTA可抑制高溫所誘導的靈芝酸產生，證明細胞外鈣離子對於熱刺激下靈芝酸生合成扮演重要角色，實驗進一步利用抑制胞器內鈣離子釋放的藥劑 (LiCl、neomycine)，證實胞器內鈣離子的釋放不在此過程扮演重要角色。另外利用鈣離子下游訊息傳遞蛋白calmodulin、calcinuerin抑制藥劑 (cyclosporine A、chloropomazine)，證實此兩個鈣離子下游訊息傳遞蛋白不扮演重要角色。本論文另一部分研究關於揮發物質，在真菌上，細胞凋亡的產生是否與揮發性二次代謝物之生合成有關，目前則是全然未知，而先前首度證實利用aspirin處理靈芝會誘導靈芝細胞凋亡及揮發性二次代謝物的產生，而熱刺激造成的細胞凋亡是否會調控不同揮發性物質是一個重要研究。實驗利用氣相層析儀觀察熱刺激誘導靈芝細胞凋亡下，發現以高溫處理靈芝所誘導的揮發性二次代謝物與aspirin處理所誘導的不盡相同，證明不同細胞凋亡方式可以誘導不同的揮發性二次代謝物產生。Ganoderma lucidum (G. lucidum ) is a traditional Chinese medicine that has been used in Asia for a long time. Its major pharmaceutical ingredient, ganoderic acids (GA), belongs to triterpenoid and it's pharmalogical activities such as anti-cancer, anti-inflammation and liver protection has been proved. In plants, several studies indicated that fungal cell walls induced apoptosis and secondary metabolite production. However, it remains unknown for regulation of apoptosis and the secondary metabolites biosynthesis. This study investigates the regulation of heat-induced apoptosis and GAs biosynthesis by cell membrane fluidity and calcium signaling. Our results showed that intracellular calcium was increased after heat stress by using calcium fluorescent dye (Fluo-3AM). 50 mM EGTA pretreatment reduce intracellular calcium concentration caused by heat treatment. By HPLC analysis, our results showed that 50 mM EGTA reduced heat-sress induced GAs production. These results indicated that extracellular calcium ions is involved in the regulation of GAs biosynthesis under heat stress. Our data also showed that calcium signaling proteins, calmoduiln and calcinurein, were not involved in heat-stress induced GAs biosynthesis by using inhibitor of calmoduiln and calcinurein, cyclosporine A and chloropomazine, respectively. In addition, we proved that calcium released from organelles was not involved in heat-stress induced GAs biosynthesis. Another part of this thesis investigate the volatile compounds in G. lucidum. Whether apoptosis regulates volatile compounds biosynthesis is unknown. Previous study indicated that aspirin induced apoptosis and volatiles production in G. lucidum. Our results showed that heat stress induced different profile of volatiles production as compared with aspirin treatment. These results indicate apoptosis regulates volatile biosynthesis in fungi.中文摘要 i Abstract ii 目錄 iii 圖表目錄 vii 1. 前言 1 1.1靈芝三萜類成分結構與藥理特性 1 1.1.1萜類化合物簡介 1 1.1.2靈芝三萜類化合物生合成機制 1 1.1.3靈芝三萜類化合物藥理活性 2 1.2細胞凋亡 3 1.2.1細胞凋亡簡介 3 1.2.2動物的細胞凋亡 3 1.2.3植物的細胞凋亡 4 1.2.4真菌的細胞凋亡 4 1.2.5細胞凋亡與二次代謝產物的關係 5 1.3熱刺激 5 1.3.1 熱刺激簡介 5 1.3.2 熱刺激與細胞凋亡的關係 6 1.3.3 熱刺激對訊息傳遞的影響 6 1.4鈣離子訊息傳遞 7 1.5揮發性成分 8 1.6實驗目的 9 2.實驗設計與材料方法 10 2.1實驗設計 10 2.2實驗試劑與儀器設備 11 2.2.1實驗試劑 11 2.2.2實驗儀器與設備 12 2.2.3實驗菌株 13 2.3實驗方法 14 2.3.1靈芝酸代謝物分析 14 2.3.2細胞內鈣離子濃度測定 15 2.3.3 固相微萃取 ( SPME ) 試驗 16 2.3.4 統計方法 16 3. 實驗結果 17 3.1改變細胞膜流動性對熱刺激造成靈芝菌絲乾重以及靈芝酸產量變化之影響 17 3.1.1降低細胞膜流動性藥物Dimethyl sulfoxide (DMSO) 對菌絲乾重的影響 17 3.1.2降低細胞膜流動性藥物DMSO對總靈芝酸和靈芝酸24產量之影響 17 3.1.3提升細胞膜流動性藥物 (benzyl alcohol) 對菌絲乾重的影響 18 3.1.4提升細胞膜流動性藥物 (benzyl alcohol) 對總靈芝酸和靈芝酸24產量之影響 19 3.2改變細胞內鈣離子訊息傳遞對熱刺激造成靈芝菌絲乾重以及靈芝酸產量變化之影響 19 3.2.1降低細胞內外鈣離子流入細胞內的藥物 (EGTA) 對菌絲乾重的影響 19 3.2.2 EGTA對總靈芝酸和靈芝酸24產量之影響 20 3.2.3胞器內鈣離子釋放抑制劑neomycin對菌絲乾重的影響 21 3.2.4胞器內鈣離子釋放抑制劑neomycin對總靈芝酸和靈芝酸24產量之影響 21 3.2.5胞器內鈣離子釋放抑制劑LiCl對菌絲乾重的影響 22 3.2.6胞器內鈣離子釋放抑制劑LiCl對總靈芝酸和靈芝酸24產量之影響 23 3.2.7鈣離子下游蛋白calcineurin抑制劑 (cyclosporine A) 對菌絲乾重變化之影響 23 3.2.8 cyclosporine A 對總靈芝酸和靈芝酸24產量之影響 24 3.2.9鈣離子下游蛋白calmodulin抑制劑chloropomazine對菌絲乾重的影響 24 3.2.10 chloropomazine對總靈芝酸和靈芝酸24產量之影響 25 3.3熱刺激下以EGTA處理細胞內鈣離子濃度改變 26 3.4熱刺激誘導靈芝細胞凋亡揮發性二次代謝物 26 4.討論 28 5. 參考文獻 3