臺灣博碩士論文加值系統

丹蒽醌（Danthron; 1,8-dihydroxyanthraquinone），是天然中草藥大黃（蓼科）中的主要成分之一，其生物活性曾經被報導，其中包括了強大的抗腫瘤活性。對於影響蛋白質功能，丹蒽醌被視為一種酪氨酸激酶抑製劑（tyrosine kinase inhibitor）。丹蒽醌不僅能抑制腫瘤細胞酪氨酸激酶信號轉導，增殖和轉移能力的，但誘癌細胞誘發細胞凋亡，包括肺癌和乳腺癌等。此外，丹蒽醌也抑制人類的子宮頸癌上皮癌，白血病，肝癌，卵巢癌，食道癌和腦膠質瘤細胞。本研究的目的是調查丹蒽醌對人類腦多形性膠質母細胞株8401株誘導細胞凋亡的機制。
在這項研究中，進行丹蒽醌誘導GBM 8401細胞的細胞毒性影響的研究。使用流式細胞儀確定細胞存活。根據這項研究的結果，丹蒽醌對於GBM 8401細胞的存活具有顯著劑量和時間依賴特性，且顯示G0/G1期停滯和sub-G1發生。利用顯微鏡觀察、DAPI染色和彗星試驗，証明丹蒽醌也造成了明顯的細胞形態損傷。流式細胞儀檢測，丹蒽醌可能影響細胞內活性氧物質（ROS）的大量產生，並促進由內質網（ER）釋出鈣和降低線粒體膜電位，造成癌細胞損傷，誘發癌細胞產生細胞凋亡機制。西方墨點法分析顯示，丹蒽醌在GBM 8401細胞會活化caspase-8、caspase - 9，和caspase - 3。預處理使用caspase-8 抑制劑 （Z-IETD-FMK）、caspase-9 抑制劑（Z-LEHD-FMK）和caspase-3 抑制劑（Z-DEVE-FMK）大大增加了GBM 8401細胞存活意味著丹蒽醌活化caspase的活性。由西方墨點法可觀察到，caspase-3、caspase-8和caspase-9，Bax、Bid、AIF、Endo G和PARP蛋白量增加而Bcl-2和Bcl-xL蛋白量降低可能導致細胞凋亡。丹蒽醌誘發細胞凋亡的透過caspase依賴和非依賴的途徑。
研究證明丹蒽醌透過粒線體相關、caspase依賴和非依賴的途徑誘發細胞毒性與凋亡，未來丹蒽醌可能作為化學藥劑作用於人類腦癌上。

Danthron (1,8-dihydroxyanthraquinone), one of component from Rheum palmatum L. (Polygonaceae), is an effective component of nature Chinese herb medicine and has been shown several biological activities inclusive of with a powerful anti-tumor activity. For the effects on protein function, Danthron is considered as a tyrosine kinase inhibitor. Danthron can not only inhibit tyrosine kinase signal pathway, proliferation and metastasis ability of cancer cells but induce apoptosis of cancer cells, including lung cancer and breast cancer etc. In addition, Danthron also inhibits human cervical epitheliod carcinoma, leukemia, hepatoma, ovarian carcinoma, esophageal carcinoma and glioma cells. The aim of this study is to investigate the mechanisms by danthron for the induction of apoptotic potential on human brain glioblastoma multiforms GBM 8401 cell line.
In this study, Danthron -induced cytotoxic effect of GBM 8401 cells were investigated. For determining cell viability, the flow cytometric analysis was used. According to the results of this study, Danthron showed a marked dose- and time-dependent inhibition of GBM 8401 cell viability and revealed G0/G1 phase arrest and sub-G1 occurrence. Danthron also caused a marked cell morphology damage proved by microscopic examination, DAPI staining and comet assay. Examined by flow cytometry, Danthron could affected on the production of a large number of intracellular reactive oxygen species (ROS) and promoted that calcium release from endoplasmic reticulum (ER) and on the decrease of the level of mitochondria membrane potential to damage cancer cells and then cause the activation of apoptosis mechanism in cancer cells. Western blotting analysis also showed the activation of initiator caspase-8 and caspase-9, and executor caspase-3 in GBM 8401 cells. Pretreatment of a caspase-8 inhibitor (Z-IETD-FMK), caspase-9 inhibitor (Z-LEHD-FMK) and caspase-3 inhibitor (Z-DEVE-FMK) significantly increased the viable of GBM 8401 cells implied that the participations of caspases. Western blots indicated that the protein levels of Caspase-3, -8, -9, Bax, Bid, AIF, Endo G, and PARP that were increased and Bcl-2, Bcl-xLthat were decreased may have led to apoptosis. Danthron induced apoptosis through the intrinsic caspase-dependent and -independent pathways in GBM 8401 cells.
This research has demonstrated that Danthron induces cytotoxicity and apoptosis in GBM 8401 cells through mitochondria-related, caspase-dependent and -independent pathways. Danthron may be further evaluated as a chemotherapeutic agent for human brain cancer

誌謝 I
中文摘要 II
英文摘要 III
目錄 V
圖目錄 VIII
表目錄 X
第一章 前言 1
1.1台灣腦瘤流行病學統計 2
1.2腦 3
1.3腦瘤 3
1.4腦瘤治療 4
1.5丹蒽醌 4
1.6細胞週期 5
1.7細胞凋亡 6
1.8粒腺體膜電位、鈣離子與細胞凋亡之關係 11
1.9活性氧化物與細胞凋亡 12
第二章 實驗目的 14
第三章 研究材料與方法 15
3.1細胞株來源 15
3.2藥品試劑 15
3.3丹蒽醌（danthron）配製 15
3.4細胞冷凍保存方法 15
3.5冷凍細胞解凍之方法 15
3.6繼代培養 16
3.7細胞分盤 16
3.8細胞存活分析 16
3.9細胞形態觀察 16
3.10細胞週期與細胞凋亡之分析 17
3.11利用DAPI染色來偵測細胞凋亡 17
3.12彗星試驗來偵去氧核糖核酸的損傷 18
3.13細胞內活性氧物質分析 18
3.14粒線體膜電位分析 18
3.15鈣離子濃度分析 19
3.16西方墨點法分析 19
3.17caspase-3、caspase-8和caspase-9活性分析 20
3.18以Real-time PCR 分析caspase-8 和caspase-9 的mRNA 含量 20
3.19統計分析 21
第四章 研究結果 22
4.1丹蔥醌對人類多型性神經膠母細胞瘤細胞株存活率的影響 22
4.2丹蔥醌對人類多型性神經膠母細胞瘤細胞株細胞形態的影響 22
4.3丹蔥醌對人類多型性神經膠母細胞瘤細胞株細胞週期與細胞凋亡的影響 22
4.4利用DAPI染色來檢測丹蔥醌誘發人類多型性神經膠母細胞瘤細胞株之細胞凋亡 23
4.5利用彗星試驗來檢測丹蔥醌誘發人類多型性神經膠母細胞瘤細胞株之去氧核糖核酸的損傷 23
4.6丹蔥醌對人類多型性神經膠母細胞瘤細胞株產生活性氧化物的影響 24
4.7丹蔥醌對人類多型性神經膠母細胞瘤細胞株粒線體膜電位改變影響 24
4.8丹蔥醌對人類多型性神經膠母細胞瘤細胞株造成鈣離子釋出的影響 24
4.9利用西方墨點法來檢測丹蔥醌對人類多型性膠母細胞株蛋白質的變化 25
4.10丹蔥醌對人類多型性神經膠母細胞瘤細胞株細胞上caspase-3、-8和-9活性的影響 25
4.11利用Real-Time PCR來檢測丹蔥醌對人類多型性神經膠母細胞瘤
細胞株之caspase-8和caspase-9 mRNA的表現 26
第五章 討論與結論 27
參考文獻 33
圖表與圖表說明 39

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