靈芝屬的真菌因已被證明具有抗腫瘤及抗老化等諸多藥理作用而被廣泛注意，在本研究，我們從新日本靈芝水萃液中分離溶於酒精的成分，以GN-ES表示，研究GN-ES對肝癌細胞(SK-Hep-1, Hep G2)及正常細胞(Rat hepatocyte, HUVEC)的增生抑制效用。實驗結果顯示，GN-ES對肝癌細胞有較顯著的增生抑制，GN-ES處理24小時的IC50劑量分別為SK-Hep-1 (0.375 mg/ml)，Hep G2 (1 mg/ml)，Rat hepatocyte (> 1 mg/ml))，HUVEC (> 1 mg/ml)), 此種選擇性增生抑制現象，可能是導因於GN-ES誘發肝癌細胞週期停滯在S期有關。西方墨點法實驗更進一步證明細胞週期停滯在S期可能是透過GN-ES增加肝癌細胞p21及p53蛋白表現所致。此外，在細胞凋亡實驗結果亦發現GN-ES會增加sub-G1期肝癌細胞的比例，GN-ES亦會增加肝癌細胞活性氧的生成及誘發粒線體膜電位下降，從Bax/Bcl-2 比值增加、caspase-9裂解蛋白增加，更進一步顯示GN-ES是透過活化粒線體調控路徑而導致肝癌細胞凋亡，綜合上述，我們推論GN-ES可能是透過阻斷細胞週期及誘發細胞凋亡來抑制肝癌細胞增生。

The genus Ganoderma has attracted considerable attention because it has been demonstrated to possess diverse pharmacological effects, such as, anti-tumor, anti-aging activity. In this study, the anti-proliferative effects of GN-ES, a ethanol-soluble fraction isolated from Ganoderma neojaponicum (GN) water extract, on human hepatoma cell lines (SK-Hep-1, Hep G2) and normal cells (Rat hepatocyte, HUVEC) were examined. Under our experimental conditions, GN-ES was found to elicit a more serious growth impediment on hepatoma cells (IC50 = 0.375 mg/ml (SK-Hep-1) and 1 mg/ml (Hep G2)) than normal cells (IC50 > 1 mg/ml) for 24 h treatment. This selective growth inhibition effect was partially attributed to cell cycle S arrest for GN-ES treated hepatoma cells. Western blotting data further demonstrated that the up-regulated p21and p53 may be the culprits for GN-ES induced cell cycle S arrest. In addition, GN-ES was also found to elicit a concentration-dependent increase in sub-G1 fraction in hepatoma cells. GN-ES-treated cells also displayed transient increase of ROS during the earlier stage of the experiment, followed by the disruption of mitochondrial transmembrane potential (ΔΨm). The increased Bax/Bcl-2 ratio and cleaved caspase-9 up-regulation further revealed that apoptosis induced by GN-ES was through mitochondria-associated apoptotic pathway. Taken together, the mechanisms of anti-proliferative activity of GN-ES in hepatoma cells may be proceed by the combined effects of the cell cycle blockage and apoptosis induction.

論文審定書+i
致謝+ii
中文摘要+iii
英文摘要+iv
縮寫表+viii
第一章 文獻回顧+1
第一節 肝癌+1
第二節 細胞週期+1
第三節 細胞凋亡+2
第四節 活性氧(Reactive Oxygen Species, ROS)和細胞凋亡的關係+3
第五節 靈芝介紹+4
第六節 研究動機與目的+5
第二章 研究方法+6
第一節 儀器及藥品+6
第二節 實驗方法+8
第三章 結果+12
第一節 GN-ES的選擇性細胞毒性試驗+12
第二節 GN-ES對於肝癌及正常細胞細胞週期及凋亡誘發情形+12
第三節 GN-ES誘發肝癌細胞過氧化物質產生情形+13
第四節 GN-ES對肝癌細胞粒線體膜電位的影響+13
第五節 GN-ES對肝癌細胞株細胞週期調控蛋白質表現的影響+13
第六節 GN-ES對肝癌細胞株Bax, Bcl-2蛋白表現的影響+14
第七節 GN-ES對肝癌細胞株Caspase-9活化情形+14
第四章 結論與討論+15
參考文獻+17
圖次+21
附錄一+33
附錄二+34
附錄三+35
附錄四+36
附錄五+37
附錄六+38

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