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研究生:謝登恩
研究生(外文):Den-En Shieh
論文名稱:大黃素抗肝癌機制與黃芩苷抗氧化作用及抗血癌機制之研究
論文名稱(外文):Studies of the mechanism on emodin-induced apoptosis in Hep/G2/C3A cells and the antioxidative effect of baicalin and its apoptotic mechanism in CCRF-CEM cells.
指導教授:林俊清林俊清引用關係
指導教授(外文):Chun-Ching Lin
學位類別:博士
校院名稱:高雄醫學大學
系所名稱:藥學研究所博士班
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:113
中文關鍵詞:大黃素黃芩苷抗氧化細胞凋亡急性淋巴性血癌細胞肝癌細胞
外文關鍵詞:AntioxidantBaicalinEmodinApoptosisHepatoma cellsAcute leukemia cells
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中文摘要 (第一篇)

許多的抗癌藥物皆具有誘發腫瘤細胞自然凋亡的作用, 但這些抗癌藥物導致細胞死亡的真正機轉至今仍是許多學者努力研究的重點。大黃素( Emodin: 1,6,8-trihydroxy-3-methyl anthraquinone )已被證實具有抗癌、抗病毒、抗多種氧化酵素等生理活性,在最近的研究中亦發現,emodin可抑制乳癌細胞tyrosine kinase的活性,而對T-淋巴細胞也具有很強的抑制作用。另外,大黃素也被報導可誘發人類肺癌細胞的自然凋亡。然而目前對於大黃素用來抗肝癌的作用機轉並未完全清楚!故本研究首先以XTT呈色分析方法,測試大黃素對三種人類肝腫瘤細胞: HepG2/C3A、PLC/PRF/5和SK-HEP-1的毒殺效果,並探討其在細胞凋亡方面可能的作用機轉。
由XTT呈色分析的實驗結果發現,大黃素對上述三種細胞人類肝癌具有很強的抑制作用,其IC50值分別為42.5、46.6和 53.1 μM。故繼之對HepG2/C3A細胞以電泳分析及流體細胞儀等實驗方法,進一步研究其毒殺肝癌細胞的機轉;根據 DNA 的裂解和sub-G1 的蓄積現象,確信大黃素對 HepG2/C3A 細胞的抑制,是經由誘發肝癌細胞走向自然凋亡以達到毒殺作用,而其自然凋亡的路徑, 經實驗得知: 大黃素具有明顯提升p53 (418.6 pg/ml)、p21(439.0 units/ml)、 Fas (6.54 units/ml)和caspase-3 (35.4 pmol/min)等相關凋亡訊息的作用。由此可知大黃素在毒殺肝癌細胞HepG2/C3A的作用機轉,可能是經由促進p53和p21的表現,造成HepG2/C3A細胞的細胞週期停滯在G2/M期,而且大黃素誘發HepG2/C3A細胞凋亡的機轉是可以經由活化p53、 p21、Fas和 caspase-3等相關蛋白質或酵素以達到其毒殺的作用,而由這些實驗的結果發現,大黃素確具有發展成為肝腫瘤化學治劑的潛力。

中文摘要 (第二篇)

最近許多的研究發現,在我們週遭環境中無所不在的氧分子以及大部分的有機體,於氧氣經由代謝以產生能量的過程中,所產生的活性氧(Reactive oxygen species, ROS),許多疾病中相當重要的病理中間產物(pathologic mediators),因此引起許多科學家廣泛的研究,並致力於預防或改善,以減輕因活性氧所導致的組織傷害。還好!在眾多的研究中發現,許多天然物確具有抗氧化的效果,故本實驗亦針對半枝蓮(Scutellaria rivularis)三種保肝活性成分:黃芩素(baicalein)、黃芩苷(baicalin)及漢黃芩素(wogonin),進行抗氧化作用機轉之研究。於實驗中,首先以黃嘌呤氧化酵素( xanthine oxidase)抑制法及細胞色素c (cytochrome c)還原比色法,測試baicalein、baicalin及wogonin對.O2- (superoxide radical)之清除能力,再以電子自旋共振法(Electron Spin Resonance, ESR)及電子補捉技術(Spin Trapping)直接測試baicalein、baicalin及wogonin對.O2- (superoxide radical)及.OH (hydroxyl radical)等自由基之補捉效應,以期解明抗氧化之作用機轉。
由於許多具有抗氧化的藥物,大多可經由誘發細胞凋亡 (apoptosis)的途徑,達成毒殺癌細胞的目的, 但細胞凋亡的進行, 常包含著許多複雜的相關訊息, 且對於不同的細胞、不同的化學藥物、其劑量的大小、或是作用時間的長短, 皆會造成不同的作用機轉。所以本實驗另外以XTT kit呈色法, 來評估baicalein、baicalin及wogonin對五種人類血癌細胞株(CCRF-CEM、K562、P3HR1、Raji、U937)的毒殺效果, 並進一步以瓊脂凝膠電泳分析及流體細胞儀探討其誘發細胞凋亡的可能性, 進而以p53,Fas,bcl-2等細胞凋亡的相關訊息蛋白及caspase-3或與粒線體相關的膜電位,和細胞質內cytochrome c的變化,探討其在細胞凋亡方面的可能作用機轉。・
由實驗結果證實baicalein、baicalin及wogonin對黃嘌呤氧化酵素( xanthine oxidase )皆具有顯著的抑制能力, 其抑制能力的大小依序為 baicalein> wogonin > baicalin, 且其IC50值分別為3.1,157.8和 215.19 μM。而抑制cytochrome c能力的大小依序為baicalin > wogonin > baicalein, 其IC50值分別為224.1, 300.1和 370.3 μM,另外由電子自旋共振法 (Electron Spin Resonance, ESR) 及電子補捉技術 (Spin Trapping) 實驗結果證實: baicalein和baicalin對hydroxyl radical(.OH)皆無清除之能力, 但對superoxide radical (.O2-)則具有很強的清除活性(SOD-like activity),其清除活性分別為baicalein:7.31×104 unit/g; baicalin:1.19 ×105 unit/g。由這些抗氧化實驗的結果發現: baicalein和baicalin具有不同的作用機轉, baicalin的抗氧化作用,主要是針對superoxide radical (.O2-)的直接補捉; 而baicalein 的主要抗氧化作用,卻表現在對xanthine oxidase 的抑制。
而在測試毒殺血癌細胞方面,由XTT呈色分析的實驗結果發現,baicalein、baicalin及wogonin對上述五種不同的人類血癌細胞株(CCRF-CEM、K562、P3HR1、Raji、U937)具有不同的毒殺效果, 其IC50值介於10.6 ~ 20.5 μg/ml間, 且以baicalin對CCRF-CEM細胞株具有最強的抑制效果, 其IC50值為 10.6 μg/ml。另由DNA 的裂解和sub-G1 的蓄積現象,確信baicalin對CCRF-CEM細胞株的生長抑制,可經由誘發血癌細胞凋亡以達到毒殺作用,而其凋亡的路徑,經實驗證實與p53和Fas的表現並無相關性, 卻與bcl-2的抑制和caspase-3的活化有明顯的關連性, 並經由對粒線體膜電位的降低和cytochrome c的釋放, 造成細胞的凋亡。而在實驗中, 針對cytochrome c 含量的測定, 我們建立了一套不用western blotting 或 ELISA kit的新實驗方法來加以測試,結果明顯發現baicalin 作用於CCRF-CEM細胞株48小時後,其cytochrome c 含量確實顯著的增加,綜合上述的實驗結果,除了讓我們對baicalin毒殺CCRF-CEM細胞的作用,及其誘發細胞凋亡的機轉有更清楚的認識外,亦提供了 baicalin做為急性淋巴性血癌化學治療劑的重要訊息。
英文摘要 (Part I)

Most of the commonly used cytotoxic anticancer drugs have been shown to induce apoptosis in susceptible cells. However, the signaling pathway of their apoptotic effects remains undefined. In this study, the cytotoxic effect of emodin on various human hepatoma cell lines was investigated. Results demonstrated that emodin exhibited strongly suppressing effect on HepG2/C3A, PLC/PRF/5, and SK-HEP-1 cells, with the IC50 value of 42.5, 46.6, and 53.1 μM, respectively. Furthermore, emodin induced apoptosis in HepG2/C3A cells was clearly verified by the appearance of DNA fragmentation and sub-G1 accumulation. Besides, HepG2/C3A cells were found to be arrested in G2/M phase after the cells were treated with 60 μM emodin for 48 h. Moreover, significant increase in the levels of apoptosis-related signals such as p53 (418.6 pg/ml), p21 (439.0 units/ml), Fas (6.54 units/ml), and caspase-3 (35.4 pmol/min) were observed in emodin treated HepG2/C3A cells. Taken together, emodin displays effective inhibitory effects on the growth of various human hepatoma cell lines and stimulates the expression of p53 and p21 that resulted in the cell cycle arrest of HepG2/C3A cells at G2/M phase. Results also suggest that emodin-induced apoptosis in HepG2/C3A cells were mediated through the activation of p53, p21, Fas/APO-1, and caspase-3. It implies that emodin could be a useful chemotherapeutical agent for treatment of hepatocellular carcinoma (HCC).

英文摘要 (Part II)

It has been indicated that the reactive oxygen species (ROS) may play an important role as pathologic medicators in various diseases. Fortunately, many herbs have been studied to exhibit a strong antioxdant activity. In this study, the antioxidant activity of baicalein, baicalin and wogonin, isolated from Scutellaria rivularis, was evaluated by modified xanthine oxidase inhibition and cytochrome c reducd methods. The results showed that the order of activity on xanthine oxidase inhibition was baicalein> wogonin> baicalin, IC50 = 3.12, 157.38 and 215.19 µM, respectively. Whereas, the activity on cytochrome c reduction was baicalin> wogonin> baicalein ( IC50 = 224.12, 300.10 and 370.33 µM, respectively). Furthermore, ESR technique was used to further confirm the direct free radical scavenging activity. Both baicalein and baicalin demonstrated a strong activity on eliminating the superoxide radical (.O2-) (baicalein: 7.31 ×104 u/g; baicalin: 1.19 ×105 u/g). The IC50 of baicalein is 2.8 fold higher than that of baicalin. However they had no significant effect on scavenging hydroxyl radical (.OH). The present results demonstrated that baicalein and baicalin posed a different pathological pathway. The antioxidative pathway of baicalin was mainly on scavenging superoxide radical whilst, baicalein was a good xanthine oxidase inhibitor.
Induction of apoptosis has been an important phenomenon involving chemotherapeutic treatment. Apoptosis is a complex cellular process, displaying multiple signal transduction controls. Use of a specific pathway may depend on cell types, chemotherapeutic drugs, dose and duration of drug exposure. Here, we demonstrated that baicalin triggered a prominent apoptotic effect in a T-acute lymphoblastic leukemia (T-ALL) cell line, CCRF-CEM, showing DNA fragmentation and cell cycle arrest at G0/G1 phase. In addition, baicalin displayed a remarkablly cytotoxic effect, with the IC50 value of 10.6 µg/ml based on XTT analysis. Several lines of evidence suggest that both p53 and Fas proteins may not mediate baicalin-induced apoptosis in CCRF-CEM cells. Instead, decline of bcl-2 mediated the occurrence of apoptosis in baicalin-treated CCRF-CEM cells and to the release of cytochrome c, the collapse of mitochondrial transmembrane potential (∆Ψm), and the activation of caspase-3. It is noticeable that we established a model, without immunoblot or ELISA kit analysis, to quantify the amount of cytochrome c in culture cells. As our results demonstrated, an observably elevation of cytosolic cytochrome c was found in baicalin-treated cells after 48 h treatment. These in vitro cell culture models elucidating the specifically cytotoxic effect on CCRF-CEM cells and the corresponding apoptotic pathway may shed light on the mechanism underlying the therapeutic effect of baicalin and intervention of chemotherapeutic agents in T-ALL.
目 錄

第一篇: 大黃素抗肝癌機制之研究

第一章 中文摘要-------------------------------------------------------------2
第二章 英文摘要-------------------------------------------------------------3
第三章 緒 論-------------------------------------------------------------4
第四章 實驗材料及方法-------------------------------------------------------6
第五章 結 果------------------------------------------------------------14
第六章 討 論------------------------------------------------------------18
第七章 參考文獻------------------------------------------------------------28
第八章 圖 表------------------------------------------------------------31

第二篇: 黃芩苷抗氧化作用及抗血癌機制之研究

第一章 中文摘要------------------------------------------------------------46
第二章 英文摘要------------------------------------------------------------48
第三章 緒 論------------------------------------------------------------50
第四章 實驗材料及方法------------------------------------------------------53
第五章 結 果------------------------------------------------------------65
第六章 討 論------------------------------------------------------------70
第七章 參考文獻------------------------------------------------------------76
第八章 圖 表------------------------------------------------------------81
第九章 附 圖---------------------------------------------------------- 109

學會及期刊發表之論文-------------------------------------------------------111
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