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研究生:孔美蘭
研究生(外文):MEI-LUNG KUNG
論文名稱:探討大豆中異黃酮素引發肝癌細胞凋亡及細胞週期調控的機制
論文名稱(外文):Study on the Mechanism of Soy Isoflavones in Regulating Cell Cycle and Apoptosis of Hepatoma Cells Lines
指導教授:張基隆張基隆引用關係
指導教授(外文):KEE-LUNG CHANG
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:81
中文關鍵詞:異黃酮素
外文關鍵詞:Isoflavones
相關次數:
  • 被引用被引用:1
  • 點閱點閱:345
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:3
由流行病學調查指出,在亞洲地區,大豆食品的攝取與降低乳癌、大腸直腸癌、前列腺癌及膀胱癌等發生率有關,但相關的研究在肝癌中尚未有完整的報導。故本研究主要以三種大豆雌激素異黃酮素(phytoestrogen isoflavones)包括Genistein、Daidzein及Biochanin A來探討其對五株肝癌細胞珠HepG2、Hep3B、HuH7、PLC及HA22T之生長抑制作用、細胞凋亡調控以及針對Genistein在HepG2細胞週期的影響。
研究結果發現Genistein及Biochanin A對五株肝癌細胞株有細胞生長抑制作用,而且與劑量比成正相關,而Daidzein則在高濃度(>40 μg/ml)的情況下對五株肝癌細胞株具有選擇性的生長抑制作用。在探討細胞凋亡方面,由西方點墨法實驗中我們發現抗凋亡蛋白Bcl-2及Bcl-XL的表現有下降的現象,此外細胞凋亡相關酵素Procaspase-3能被分解而活化,使Caspase-3酵素的受質PARP(poly(ADP-ribose)polymerase)裂解產生85 KD片段。
另外在流式細胞分析儀(Flow cytometry)實驗分析中也發現,Genistein可引發肝癌細胞株HepG2之細胞週期停滯在G2/M期,而針對此結果,我們做了進一步的探討。其結果發現,當以不同濃度的Genistein處理HepG2細胞24小時後,調控G2/M期進行的蛋白Cyclin B1的表現並不受到影響;然而由Cdc2 kinase活性及蛋白表現量皆受到抑制。此外,我們也針對Cyclin B1/Cdc2的上游蛋白Cdc 25C磷酸酶(Cdc25C phosphatase)、Chk2、p53及ATM等蛋白活性及表現情形來研究。其結果發現,Cdc25C磷酸酶的表現會受到Genistein的抑制;而Chk2、p53蛋白的表現及ATM蛋白活性則有增加的現象;另外,p53蛋白下游的p21waf1/cip1亦有高度表現(over-expression)的情形。綜合上述結果,我們認為Isoflavones能抑止肝癌細胞生長,影響抗凋亡蛋白Bcl-2及Bcl-XL的表現並促使細胞凋亡。而且經由ATM蛋白抑制劑-咖啡因(Caffeine)及Genistein調控肝癌細胞HepG2之G2/M期進行之研究,我們得知,以Caffeine單獨處理細胞時,會明顯抑制ATM下游蛋白分子Chk2、p53及Cdc2-p的表現,而當Caffeine及Genistein共同處理細胞時,則發現Genistein能逆轉Caffeine對Chk2、p53蛋白及Cdc2 kinase所造成的影響。因此我們推論,Genistein誘導HepG2細胞週期停滯在G2/M期,可能是透過ATM訊息傳遞路徑來進行。

Epidemiological studies have shown that soy foods consumption was associated with the reduction of the risk of breast, colon, prostate, and bladder cancers. However, few studies have investigated the effects of the soy foods on hepatocellular carcinoma. The aim of this study was to investigate three soy Isoflavones, including Genestein, Biochanin-A, and Daidzein, on cell growth, the cell cycle and apoptosis induction in the human hepatoma cancer cell lines, HepG2, Hep3B, Huh7, PLC, and HA22T. Results showed that Genestein and Biochanin-A inhibited growth of all five lines in a dose-dependent manner, whereas Daidzein has selective effects. Activation of Caspase 3 and cleavage of the Caspase 3 substrate, poly (ADP-ribose) polymerase, was seen in hepatoma cells after 24 hours exposure to soy Isoflavones. In addition, Isoflavones cytotoxicity corrected with down-regulation of Bcl-2 and Bcl-XL expression.
Our laboratory has recently reported that Genistein arrests hepatocellular carcinoma cells in G2/M phase. In this study, we further investigate the cell cycle’s regulators. Results showed that Genistein decreased the activity of Cdc2 and increased the phosphorylation of Cdc2, whereas the Cyclin B expression was not effected. In addition, Genistein enhanced the expression of the cell cycle inhibitor p21waf1/cip1 and tumor suppressor p53 Moreover, Cdc25C phosphatase was reduced. From these results, we conclude that genistein arrest HepG2 cells at G2 phase might be through decreasing in Cdc25C phosphatase resulting in Cdc2 activity decrease as well as through induction of p21waf1/cip1 which was interaction with Cdc2. We also found Genistein activated the checkpoint kinase Chk2; and activated ATM activity. All these effect of Genistein would be impaired by caffeine which is a n inhibitor of ATM kinase. Taken together, the Genistein’s effect of G2 arrest in HepG2 cells by increasing phosphorated/inactivated Cdc25C phosphatase could be mediated, even in part, through ATM-dependence of Chk2 activation.

目 錄
中文摘要--------------------------------------------------------------------1-2
英文摘要--------------------------------------------------------------------3-4
緒論--------------------------------------------------------------------------5-14
研究材料與方法-----------------------------------------------------------15-25
結果--------------------------------------------------------------------------26-36
討論--------------------------------------------------------------------------37-44
參考文獻--------------------------------------------------------------------45-61
圖表--------------------------------------------------------------------------62-81

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