中文摘要
癌症是目前造成人類致死率最高的疾病，因此全世界許多醫藥專家致力於尋求有效的抗癌藥物。由於紫杉醇成功的開發成為有效的抗癌藥物，由天然物中萃取抗癌化合物的研究趨勢，已成為日本、德國及美國等科學先進國家的藥廠及研究者爭相競爭的方向。中藥科學化的研究亦為目前國內生物醫學界努力的目標。肝癌為台灣地區發生率最高、死亡率最高的癌症，本論文以人類肝癌細胞株為研究模式，探討中藥黃芩中之主要成分之一的黃芩苷元 ( Baicalein ) 抑制癌細胞生長及誘導癌細胞凋亡之分子機制。由實驗結果中得知，黃芩苷元可抑制人類肝癌細胞株 ( HepG2、Hep3B、Huh-7 ) 之生長，且呈現時間、劑量之正相關性。以黃芩苷元處理Hep3B細胞各一、二、三、四天後，以西方墨點法分析細胞內蛋白質的表現量，結果發現處理黃芩苷元三天之Hep3B細胞中之Bcl-XL蛋白表現量逐漸減少，且caspase-8及caspase-3分解成活化片段，PARP [ poly( ADP-ribose ) polymerase]也被裂解成一89 kD的片段。但經黃芩苷元處理的Hep3B細胞中Fas和Fas-L之表現並沒有明顯變化，顯示此凋亡路徑並無活化。我們進一步探討黃芩苷元誘發肝癌細胞凋亡時，與死亡相關的訊息傳遞分子MAPK家族蛋白之變化。結果顯示黃芩苷元會促使 MEK、ERK、JNK和c-Jun蛋白的表現量增加，且磷酸化的MEK，ERK及JNK也有明顯增加的現象。綜合以上之結果，我們可知黃芩苷元誘導之細胞凋亡，是一種不需經細胞膜死亡受體Fas /Fas-L之活化，而是經由Bcl-XL蛋白量的減少，造成粒線體膜功能的缺失，誘發了caspase-3的活化導致細胞的凋亡。而黃芩苷元對細胞造成的死亡壓力 ( stress ) 可能是經由MAPK家族的JNK及ERK的訊息傳遞路徑。

Abstract
Baicalin, a flavonoid, isolated from the dried root of Scutellaria baicalensis Georgi ( Huang Qin ), is widely used in the traditional Chinese herbal medicine for its anti-inflammatory, anti-proliferative and anti-cancer effects. A resent study has demonstrated that Baicalein can induce apoptotic cell death in human cancer cells. In this study, we investigate the mechanism of Baicalein-induced apoptosis in human hepatoma cells. The results showed that Baicalein caused a time- and dose-dependent cell death in three tested human hepatoma cell lines. In Hep3B cells, Baicalein-mediated growth inhibition was accompanied by the down-regulation of Bcl-XL, and activation of caspase-8 and caspase-3, while had no effect on the level of Bak and Fas proteins. Moreover, phosphorylated MEK、ERK and JNK proteins were increased after Baiclaein treatment. These results suggest that Baicalein mediated the apoptosis of Hep3B cells via a Fas-independent and mitochondrial-controlled manner, and the JNK、 ERK activation cascade might be responsible for this death signal.

目錄
壹、 摘要
中文摘要 1
英文摘要 3
貳、 前言 4
參、 材料與方法
一、細胞株來源 14
二、試劑種類及來源 14
三、細胞培養液與藥劑的配製 17
四、細胞株之培養 18
五、細胞生長之測定 18
六、蛋白質之定性及定量分析 19
肆、 結果
一、黃芩苷元對人類肝癌細胞株生長的影響 23
二、黃芩苷元誘導人類肝癌細胞凋亡過程中相關分子的表現…..24
伍、 討論
一、黃芩苷元對肝癌細胞株之生長抑制作用 30
二、黃芩苷元誘導細胞凋亡之分子機制 31
三、黃芩苷元誘導細胞凋亡時之訊息傳遞路徑 37
陸、 結論 40
柒、 參考文獻 41
捌、 圖示說明 54

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