| 摘要: | 絞股藍皂?誘導人類大腸癌細胞株 (Colo 205) 的細胞週期停滯及細胞凋亡 研究生:呂孔文 指 導 教 授 :陳榮洲副教授 中國醫藥大學中國醫學研究所 絞股藍是中藥的一種，臨床研究顯示絞股藍的粗抽物有不同的藥 理作用，包括抗癌，提高免疫能力等，絞股藍皂?是絞股藍主要的 粗抽物，且有報告指出，可誘導人類大腸癌細胞株凋亡。然而，絞股 藍皂?誘導細胞凋亡的分子機轉尚不清楚；本實驗，我們發現 在存活率方面，絞股藍皂?作用於人類大腸癌細胞株(Colo 205)有時 間和濃度的依賴性。更進一步，我們藉由觀察細胞形態的改變，DAPI螢光染色及Comet assay來研究gypenosides對Colon 205 產生細胞凋亡的影響。另一方面，DNA的凝膠電泳證明了絞股藍皂?能誘導人類大腸癌細胞走向凋亡；流式細胞儀能分析證明絞股藍皂?能誘導人類大腸癌細胞生長停滯於sub-G1期。本實驗並利用西方墨點法了解細胞週期與細胞凋亡間的相關蛋白。數據顯示，絞股藍皂?誘導人類大腸癌細胞株(Colo 205)細胞週期停止在G0/G1 期是透過 p21、 p27、 p16、 p15的表現量增加， Cyclin D、 Cyclin E、 CDK4(6)、 CDK2的表現量減少。絞股藍皂?誘導細胞凋亡是透過 p53、 BAX、 caspase 9、 caspase 3、 cytochrome c的表現量增加，BcL-2、 BcL-XL的表現量減少。並藉由流式細胞儀分析結果 Ca2+、ROS (Reactive Oxygen Species)的表現量增加，MMP(粒腺體膜電位)的表現量減少。這些結果顯示絞股藍皂?誘導大腸癌細胞株走向細胞凋亡為透過粒腺體路徑或氧化還原路徑。 關鍵字：絞股藍皂?，人類大腸癌細胞株(Colo 205)。; Gypenosides induce cell cycle arrest and apoptosis in human colon cancer cells line (Colo 205) Kung-Wen Lu Major professor： Jung-Chou Chen Institute of Chinese Medical Science, China Medical University Gynostemma pentaphyllum (GP) is one of the Chinese medical herb. Clinical researches indicated that the extracts of GP have various pharmacological effects including anti-cancer, enhancing immune capacity effect etc. Gypenosides(Gyps) is a major compound of Gynostemma pentaphyllum Makino and is reported to induce apoptosis in human colon cancer cells. However, the molecular mechanism underlying the gypenosides induced apoptotic process is still unclear. In the present studies, we have observed that treated Colo 205 cells with gypenosides resulted in a dose- and time-dependent decrease in cell viability. Furthermor, we found the gypenoside induced apoptosis in human Colon 205 cells by morphological changes, DAPI (4',6-diamidino-2-phenylindole) staining and comet assay. Furthermor, DNA fragmentation agarose gel electrophoresis also confirmed that gypenosides induced apoptosis in human Colo 205 cancer cell line. Flow cytometric analysis demonstrated that the appearance of the sub-G1 phase in human Colo 205 cancer cell line after treated with gypenosides.In this study,the Western blot will be used for detecting cell cycle associate protein and apoptosis. The data display that Gyps induced G0/G1 phase arrest of cell cycle in Colo 205 cells through the up-regulation of p21, p27, p16, p15 and down-regulation of Cyclin D, Cyclin E, CDK4(6), CDK2. These results suggest that Gyps induced apoptosis through the up-regulation of p53, BAX, caspase 9, caspase 3, cytochrome c and down-regulation of BcL-2, BcL-XL. Flow cytometry assays also confirmed that the level of Ca2+, ROS were increased and MMP was decreased. These results indicated that Gyps induced apoptosis of Colo 205 cells through the mitochondria-dependent pathway or redox signaling pathways. Key words： Gypenosides, Colo 205 |
