絞股藍皂是絞股藍的主要有效成分，現代藥理證實絞股藍有抗癌作用，在動物及體外實驗證明，具有明顯的抑制癌細胞作用，然而絞股藍皂抗癌作用之機轉尚無文獻報導，因此本論文為進一步闡明絞股藍抗癌的作用機制，從細胞和基因層次，探討絞股藍皂誘發人類肝癌細胞凋亡之調控機轉。實驗以人類肝癌細胞株(Huh-7，Hep3B，HA22T/VGA)，結果發現絞股藍皂皆能抑制這三株肝癌細胞生長，且絞股藍皂抑制細胞生長數，隨濃度與時間，皆有降低之效應。進一步以DAPI與TUNEL螢光染色分析法，發現細胞膜形成皺縮、細胞核染色質濃縮、細胞核碎裂和染色體DNA斷裂，確定細胞進行細胞凋亡形狀特徵。針對誘發細胞凋亡之調控路徑，在caspase家族方面，發現絞股藍皂能增加caspase-1、9、3之活性，但不影響casapas-8之活性，而且用caspase 抑制劑 (z-VAD-fmk)，發現能抑制絞股藍皂誘發caspase之活性，更確定絞股藍皂誘發caspase之路徑。絞股藍皂能誘發caspase-1、9活性，其為caspase-3之上游(upstream)，能誘發caspase-3活性增加而執行細胞凋亡，它能造成cytochrome c從粒腺體外膜上釋放至細胞質，造成具DNA修補基因體功能之PARP被分解，使染色體DNA斷裂成片斷形成細胞凋亡。在Bcl-2 家族方面，絞股藍皂能增加Bax、Bak基因表現，降低Bcl-2基因表現，但不改變Bag-1基因表現。在Fas與Fas Ligand訊號接收系統方面，結果發現絞股藍皂不影響Fas基因表現，且不影響其下游之casapase-8之活性，顯示絞股藍皂誘發細胞凋亡之調控路徑與Fas、Fas Ligand系統無關。在p53抑癌基因方面，結果發現絞股藍皂不影響P53抑癌基因表現，無論是HA22T/VGA (wild type p53)、Huh-7 (mutant pP53) 或Hep3B（完全沒有p53蛋白表現），顯示絞股藍皂誘發細胞凋亡之調控路徑與p53無關。綜合上述結果顯示，絞股藍皂誘發細胞凋亡之調控路徑，主要是由於促進細胞凋亡功能的基因Bax、Bak大量表現，且抑制細胞凋亡功能的基因Bcl-2之表現減少，促使cytochrome c從粒腺體外膜上釋放至細胞質中，使得caspase家族活化，進而誘發細胞凋亡。

Gypenosides are triterpenoid saponins contained in an extract from Gynostemma pentaphyllum Makino and reported to induce apoptosis inhuman hepatoma cells. However, the molecular mechanism underlying the Gypenosides-induced apoptotic process is unclear. To understand the molecular mechanism of Gypenosides-inducced apoptosis, we had detected the caspase cascade, Bcl-2 family, Fas/Fas Ligand system, and p53 gene regulation. In this study, we have observed that treatment with Gypenosides resulted in a dose- and time-dependent decrease in cell viability. We found that Gypenosides-induced apoptosis in human hepatoma Huh-7, Hep3B and HA22T cell lines as evidenced by morphological changes, 4’ ,6’- diamidino-2-phenylindole (DAPI) staining and in situ terminal transferase-mediated dUTP-fluorescensinnick end-labeling (TUNEL) assay. In Bcl-2 family, our data demonstrated that Gypenosides -induced apoptotic cell death was accompanied by up-regulation of Bax, Bak and Bcl-XL , and down-regulation of Bcl-2 and Bad, while it had no effect on the level of Bag-1 protein. In caspase cascade, moreover, Gypenosides treatment caused the release of mitochondrial cytochrome c to cytosol and sequential activation of caspases, including caspase-1, -9 and -3, then leading to cleavage of poly-ADP-ribose polymerase. Furthermore, the Gypenosides-induced apoptosis was markedly blocked by the broad-spectrum caspase inhibitor, z-VAD-fmk. In Fas/Fas Ligand system, our results showed that caspase-8 was not activated upon Gypenosides treatment, and Gypenosides did not affect the expression levels of Fas and Fas Ligand proteins, indicating that stimulation of Fas/Fas Ligand and activation of caspase-8 did not involve in Gyp-induced caspase-3 activation and apoptosis. In p53 gene regulation, no accumulation of wild-type p53 protein was detected upon treatment of HA22T cells with Gyp. In addition, cells lacking functional p53 (Hep3B and Huh-7 cells) exhibit a similar response to Gypenosides treatment as do cells with functional p53 (HA22T cells). These results suggest that Gypenosides-mediated up-regulation of Bax protein and apoptosis occurred independence of p53 protein.Taken together, these results suggest that treatment of human hepatoma cells with Gypenosides induced apoptosis through the up-regulation of Bax and Bak, and down-regulation of Bcl-2, release of mitochondrial cytochrome c and activation of caspase cascade.

第一章 前 言 1
第二章 文 獻 探 討 4
第一節 絞股藍與絞股藍皂 4
第二節 細胞凋亡 21
第三章 材 料 與 方 法 36
第四章 結 果 43
一、劑量效應之生長曲線(DOSE-DEPENDENT CELL VIABILITY) 43
二、時間效應之生長曲線(TIME-DEPENDENT CELL VIABILITY) 43
三、凋亡細胞核之螢光染色(DAPI AND TUNEL STAINING ASSAY) 45
四、絞股藍皂引起CASPASE活化 (GYPENOSIDES-INDUCED ACTIVATION OF CASPASES) 48
五、CASPASE 抑制劑 (Z-VAD-FMK)抑制絞股藍皂誘發細胞凋亡 (INHIBITION OF CASPASE ACTIVITY IN GYPENOSIDES-INDUCED APOPTOSIS BY CASPASE INHIBITORS) 56
六、CYTOCHROME C之釋出先於CASPASE-3活化(RELEASE OF CYTOCHROME C OCCURRED PRIOR TO THE ACTIVATION OF CASPASES) 56
七、絞股藍皂對BCL-2 家族蛋白質表現之影響(BCL-2 FAMILY PROTEIN EXPRESSION BY GYPENOSIDES) 59
八、絞股藍皂對P53抑癌基因蛋白質表現之影響(REGULATION OF P53 PROTEIN EXPRESSION BY GYPENOSIDES) 65
九、絞股藍皂對FAS基因蛋白質表現之影響(REGULATION OF FAS PROTEIN EXPRESSION BY GYPENOSIDES) 67
十、絞股藍皂對MAPK家族基因表現之影響(Regulation of MAPK system expression by Gypenosides) 67
第五章 討 論 71
第六章 結 論 78
參 考 文 獻 81
英 文 摘 要 99
謝 辭 100
附 錄 101

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