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研究生:鍾佩君
研究生(外文):Pei-Jun
論文名稱:龍葵水萃取物及多酚致肝癌細胞凋亡及機轉之研究
論文名稱(外文):SNWE and SNPE -mediated human hepatocellular carcinoma cells apoptosis and mechanism research
指導教授:王朝鐘王朝鐘引用關係
指導教授(外文):Chau-Jong Wang
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:生化暨生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:90
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龍葵,為分布於全球的耕地、花園的野生植物。已被證實具有抗氧化、抗發炎、護肝作用以及抗腫瘤之醫學功能。在中國地區,龍葵及龍葵複方更是被廣泛用於惡性腫瘤後的治療。在本篇研究中證實了龍葵水萃物對多種癌細胞具有毒性,其中以肝癌細胞HepG2對龍葵水萃取物的處理最敏感,因此我們後續將以HepG2為研究之主軸。此外我們更進一步由龍葵水萃取物中分離出多酚成分,並由MTT分析結果得知龍葵多酚也會對HepG2細胞產生毒性,且所需的劑量較龍葵水萃物低。我們接著發現龍葵水萃及多酚會誘導細胞產生核濃染、DNA片段化以及細胞週期sudG1期增多的現象,判斷龍葵水萃及多酚萃取物對HepG2細胞的毒性是引發其邁向細胞亡。且其造成細胞凋亡的機轉是增加Fas表現而促進caspase-8的切割,活化後的caspase-8則進一步活化下游caspase-3以及Bid的切割,最後導致PARP失去作用而無法修復DNA,使細胞走向凋亡。此外,我們也發現低劑量之龍葵水萃及多酚萃取物會造成HepG2細胞週期停滯於G2/ M期。除了上述的細胞實驗外,我們也在裸鼠的動物實驗結果中發現,龍葵水萃及多酚萃取物能抑制肝癌細胞形成腫瘤。綜合細胞以及動物實驗的結果,我們推測龍葵具有治療肝癌的潛力。且其功效可能是來自於其中的多酚成份,期許將來在肝癌的治療,龍葵多酚可成為肝癌患者更好的選擇。

Solanum nigrum L. is worldwide weeds of arable land, gardens. It is believed to have anti-oxidative, anti-inflammatory, hepatoprotection and anti-tumor effects. In China, it has been used in traditional folk medicine to treat different cancers. In this study, we demonstrated that the water extract of Solanum nigrum L. (SNWE) expressed cytotoxic effects to many cancer cell lines. Among them, human hepatocellular carcinoma cells (HepG2) were the most susceptible to SNWE. We also isolated the polyphenolic compounds (SNPE) form SNWE and then used MTT assay to evaluate the cytotoxicity on the HepG2 cells. The results show that HepG2 cells were more susceptible to SNPE than SNWE.
Next, we observed the effects of SNWE and SNPE in inducing apoptosis in HepG2 cells by measuring the nuclear condensation, DNA fragmentation and increased the subG1 phase ratio in cell cycle. This effect of SNWE and SNPE in HepG2 cells might be mediated via increase the expression of Fas and cleaved-caspase 8, 3 protein. Furthermore, we treatment low dose of SNWE and SNPE to HepG2 cells and resulted in significant cell cycle arrest in G2/M phases. It might be due to decrease CDC25C protein expression.
Besides in vitro effects, the antitumor efficacy of SNWE and SNPE were also confirmed in human hepatocellular carcinoma xenografts in nude mice. Thus, our data indicate that SNWE and SNPE could play an active role in mediating the cell cycle arrest and apoptosis of human hepatocellular carcinoma cells and might be potential drugs for antitumor therapy.


目錄
縮寫表 4
中文摘要 5
英文摘要 6
序論 7
肝癌簡介 7
龍葵(Solanum nigrum L.)簡介 9
多酚( polyphenol )簡介 13
細胞週期(cell cycle)簡介 18
細胞凋亡(cell apoptosis)簡介 22
研究動機 29
研究架構 30
實驗材料與方法 30
實驗材料與方法 31
實驗方法 34
結果 47
討論 57
參考文獻 63
圖表 69



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