臺灣博碩士論文加值系統

許多的研究報告指出三萜類化合物具有抗癌藥性，本實驗室先前已經證實台灣特有蕈菇類植物牛樟芝的重要化學成份之一: 三萜類化合物－去氫齒孔酸(Dehydroeburicoic acid, DeEA)，會造成神經膠原瘤細胞U87MG大量消耗ATP、細胞內鈣離子濃度增加、活化calpain，而引發細胞壞死(necrosis)。因此，本論文的主要目的是要進一步地探討DeEA造成U87MG細胞壞死路徑的分子機制，並同時研究DeEA的兩種衍生物：21號碳添加甲基的Methyl Dehydroeburicoate (M-DeEA)，以及3號碳加乙酸酯基的Dehydroeburicoic acid monoacetate (MA-DeEA)， 對神經膠原瘤細胞生長的影響。
神經膠原瘤細胞在經過三種藥物處理24小時後，細胞存活率和細胞毒性分析結果顯示DeEA、M-DeEA和MA-DeEA皆會抑制細胞的生長能力。在光學顯微鏡下觀察細胞的形態變化，發現三種藥物造成神經膠原瘤細胞不同的形態變化：DeEA造成細胞縮短呈現球狀而且破裂，M-DeEA處理的細胞也有呈現縮短但是沒有明顯細胞破裂， MA-DeEA則造成細胞呈現不規則細長形態或呈圓形漂浮的狀態。因此，我們進一步分析比較這三種藥物對U87MG 細胞生長抑制與死亡機制的差異。
藥物處理後的細胞以Annexin V/propidium iodide (PI)染色，再以流式細胞儀分析細胞死亡形式，結果顯示DeEA會造成U87MG細胞壞死，M-DeEA和MA-DeEA則是傾向增加細胞的凋亡。以AO/ EtBr染色分析細胞死亡形式，也顯示M-DeEA會增加細胞凋亡的比例。但當細胞以抑制細胞凋亡的蛋白酵素抑制劑先處理以抑制細胞凋亡，並不能明顯抑制MA-DeEA造成的細胞死亡。此外，以PI染色分析細胞週期變化時則發現MA-DeEA會促進細胞停留在G2/M期。
以免疫螢光染色偵測自噬體膜蛋白LC3B的變化，結果顯示DeEA會明顯導致細胞內自噬體的增加。以Mitotracker偵測細胞內粒線體膜電位的變化， DeEA和MA-DeEA都會降低細胞內粒線體的功能，可能因而導致細胞的死亡。我們也以免疫螢光染色觀察藥物處理後的細胞PARP和AIF的變化，結果顯示DeEA和M-DeEA會造成AIF從粒線體移位到細胞核的現象，而且DeEA還引起PARP蛋白質表現顯著增加的情形。
綜合以上結果，我們發現DeEA、M-DeEA和MA-DeEA會對神經膠原瘤細胞U87MG產生不同的生長抑制作用。DeEA造成的細胞壞死和PARP/AIF有關，M-DeEA會造成細胞偏向細胞凋亡的死亡機制，而MA-DeEA則可能會使細胞生長週期停留在G2/M時期。

Numerous studies have demonstrated the anticancer effect of triterpenoids. A previous study from our lab has shown that Dehydroeburicoic acid (DeEA), a triterpenoid isolated from Antrodia cinnamomea (known as “niu-chang-chin” in Taiwan), can induce necrotic cell death in the human glioblastoma cell U87MG via Ca2+ overload, mitochondrial dysfunction, and calpain activation. In this thesis, we aim to further examine the mechanisms of DeEA-induced cell death in U87MG cells. In addition, we explored the anti-proliferative effects of two DeEA-derived compounds, Methyl Dehydroeburicoate (M-DeEA) and Dehydroeburicoic acid monoacetate (MA-DeEA).
Cell viability and cytotoxicity assays indicated that all three compounds can inhibit the proliferation of U87MG cells. Light microscopic analyses showed that the three compounds display differential morphological effects: DeEA induced shortening round-up and cell rupture; M-DeEA also induced cell shortenting, but with no significant rupture; and MA-DeEA induced irregular cell elongation or floating round-up. We therefore moved on to examine the differential anti-proliferation and cell death mechanisms of the three compounds.
Flow cytometric analyses with Annexin-V and propidium iodide labeling showed that DeEA significantly enhanced necrotic cell death, but that both M-DeEA and MA-DeEA moderately increased apoptotic cell death. The results of AO/EtBr staining assay also supported the idea that M-DeEA induced apoptosis. Pre-treatment with the caspase inhibitor zVAD-fmk, however, failed to significantly block MA-DeEA-induced apoptotic cell death. On the other hand, cell cycle analyses with PI labeling suggested that MA-DeEA induced cell cycle arrest at the G2/M phase.
Immunofluoresence staining of LC3B showed that DeEA significantly enhanced the presence of autophagosomes, Examination of the mitochondrial membrane potential with Mitotracker demonstrated that both DeEA and MA-DeEA disrupted the function of mitochondria, which in turn may induce cell death. Immunofluoresence staining of PARP and AIF further showed that both DeEA and MA-DeEA induced the translocation of AIF from mitochondria to nucleus, and that DeEA notably enhanced the expression of PARP.
In summary, we have identified differential cell death effects in U87MG cells by DeEA, M-DeEA, and MA-DeEA: DeEA causes PARP/AIF-related necrosis; M-DeEA triggers apoptotic cell death; and MA-DeEA probably induces cell cycle arrest at the G2/M phase.

目錄.................................................................................................................................i
圖次...............................................................................................................................iv
英文縮寫表...................................................................................................................vi
中文摘要......................................................................................................................vii
英文摘要.......................................................................................................................ix
第一章 導論
1.1 牛樟芝 (Antrodia cinnamomea)....................................................................1
1.2 去氫齒孔酸 (Dehydroeburicoic acid, DeEA)...............................................2
1.3 細胞週期 (Cell cyle).....................................................................................2
1.4 細胞死亡 (Cell death)...................................................................................4
1.4.1 細胞凋亡 (Apoptosis).........................................................................4
1.4.2 細胞自噬 (Autophagy).......................................................................6
1.4.3 細胞壞死 (Necrosis)...........................................................................8
1.5 神經膠原瘤細胞 (Glioblastoma)................................................................10
1.6 研究目的 (Aim of the study)......................................................................11
第二章 材料與方法
2.1 細胞培養 (Cell culture)..............................................................................13
2.1.1 人類神經膠原瘤細胞 (U87MG cell line)........................................13
2.1.2 人類神經膠原癌細胞株 (GBM 8401 cell line)...............................13
2.1.3 繼代培養 (Subculture)......................................................................13
2.2 藥物處理與加入 (Drugs treatment)...........................................................14
2.3 細胞存活分析 (MTT cell viability assay)..................................................14
2.4 倒立式顯微鏡照相 (Phase contrast photo)................................................15
2.5 細胞毒性檢測乳酸去氫酶 (LDH releasing assay)....................................15
2.6 流式細胞儀分析 (Annexin V and propidium iodide double stain)............16
2.7 細胞週期測定 (Propidium iodide stain).....................................................17
2.8 凋亡蛋白酵素抑制劑測試 (Caspase inhibitor test, zVAD-fmk)...............17
2.9 Acridine orange和Ethidium bromide染色 (AO/EtBr stain)......................18
2.10 細胞螢光染色 (Flourence stain).................................................................18
2.11 西方墨點法 (Western blotting)...................................................................19
2.12 數據統計分析 (Software and data analysis)...............................................20
第三章 實驗結果
3.1 利用MTT assay來檢測DeEA以及兩種衍生物是否會影響細胞生長....21
3.2 以倒立式顯微鏡觀察細胞經過DeEA及其衍生物處理後的形態變化...21
3.3 利用LDH assay觀察細胞經過三種藥物處理24小時，細胞破裂死亡的 狀況..............................................................................................................23
3.4 以流式細胞儀分析annexin V and propidium iodide雙重染色細胞，測定 三種DeEA藥物是否引發細胞的死亡途徑...............................................23
3.5 以流式細胞儀分析propidium iodide染色細胞，檢測三種藥物是否引發 細胞週期的變化..........................................................................................24
3.6 以Acridine orange和Ethidium bromide染色，檢測三種藥物對於U87MG 細胞膜完整性的影響..................................................................................25
3.7 以Phalloidin標示filamentous actin，檢測三種藥物對於細胞生長形態和 移動能力的影響..........................................................................................26
3.8 以MitoTracker標示細胞內粒線體的分布情形，檢測三種藥物對於細胞 能量利用的影響..........................................................................................27
3.9 以anti-LC3B抗體染色，檢測三種藥物的處理是否會引發細胞自噬......27
3.10 以抗體進行免疫染色，檢測三種藥物的處理是否會影響PARP-1會導致 粒線體釋放AIF並且移位到細胞核..........................................................28
3.11 以西方墨點法檢測PARP蛋白在控制組是否有被caspase切割的細胞凋 亡特徵..........................................................................................................29
3.12 利用LDH assay分析細胞凋亡蛋白酵素抑制劑是否會影響三種藥物所 引起之細胞反應..........................................................................................29
3.13 以西方墨點法檢測細胞週期素蛋白Cyclin A和Cyclin B1在不同神經膠 原瘤細胞株控制組的表現情形..................................................................29
3.14 以抗體進行免疫染色，檢測MA-DeEA的處理是否會引發神經膠原瘤細 胞進行細胞再分化......................................................................................30
第四章 討論
4.1 DeEA及其衍生物M-DeEA和MA-DeEA造成神經膠質瘤細胞不同生長 抑制效果的差異..........................................................................................31
4.2 DeEA及其衍生物M-DeEA和MA-DeEA造成不同的細胞死亡模式.....33
4.3 DeEA所產生的細胞壞死和MA-DeEA導致的細胞週期停滯機制.........35
4.4 問題與檢討..................................................................................................36
4.5 結論..............................................................................................................37
實驗圖表......................................................................................................................38
參考文獻......................................................................................................................56

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