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研究生:王致為
研究生(外文):Jhih-Wei
論文名稱:紫花地丁與葉下珠抑制肺癌細胞侵入及移動能力之機制探討
論文名稱(外文):Study of the inhibitory effects and its mechanism of viola yedoensison and phyllanthus urinaria on invasion and migration of lung cancer cells
指導教授:謝易修謝易修引用關係
指導教授(外文):Yih-Shou Hsieh
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:103
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癌細胞的轉移往往是惡性腫瘤的重要特徵之一,並且也是在臨床治療上最難以根治的。因此,在很多研究中,大都是以針對癌細胞的轉移過程,開發新藥或是利用天然藥物處理是否會影響癌細胞的轉移。目前已有多種中藥已被確認具有抗發炎,抗過敏及抗癌的活性,且其中具有生物活性的有效成份,部分已被單離並經分析鑑定。中藥對癌症所引起的諸多症狀的治療能有所助益,已是眾所皆知的事實,而中藥的抗癌有效成份之作用機轉,常為有效地促使癌細胞凋亡及抑制癌細胞增生,但對其抑制侵襲及轉移作用和其詳細機制方面的研究還很少。因此,本研究選用一株具有高度轉移能力的人類肺癌細胞:A549,處理紫花地丁與葉下珠的萃取物來探討對於癌細胞侵入能力的影響。首先藉由modified Boyden chamber invasion assay,發現紫花地丁與葉下珠萃取物具有抑制A549侵入的能力,而且不具有細胞毒性。而當癌細胞轉移時常常會伴隨著細胞外基質的分解及細胞移動能力的改變,在gelatin zymography與casein zymography assay中也發現到它們可以抑制A549人類肺癌細胞的MMP-2及u-PA的表現。因此我們再利用RT-PCR及Western blot發現紫花地丁與葉下珠萃取物會抑制A549人類肺癌的蛋白酶,及改變蛋白酶抑制劑的表現。除此之外,我們也發現紫花地丁萃取物具有抑制p38磷酸化、細胞核內NF-kappaB的表現及其DNA binding的活性;而葉下珠萃取物具有抑制JNK及Akt磷酸化、細胞核內AP-1的表現及其DNA binding的活性。此外,還利用A549-bearing BALB/c mice與LLC-bearing C57BL/6 mice的動物模式,觀察到紫花地丁與葉下珠萃取物對於腫瘤生長、最終腫瘤重量與腫瘤轉移至肺臟數目都有一個明顯的抑制情形。綜合以上結果,紫花地丁與葉下珠萃取物可能是透過抑制MAPK pathways中蛋白的磷酸化及轉錄因子的活性,進而抑制癌細胞轉移相關的蛋白酶及影響蛋白酶抑制劑來達到抑制癌症轉移的能力。

The metastasis of cancer is a vital trait in malignance with extreme difficulties in early diagnosis and therapeutic management. Therefore, the development of new remedies or the utilization of natural medicines targeted at metastasis has been interested and studied extensively. At present many kinds of Chinese medicinal herbs have the anti- inflammation, anti-allergic and anti-cancer activeness. Part of active principles had to be isolated and identified. Recently, Chinese medicinal herbs have been reported to have various anti-carcinogenesis properties, including inducing cell apoptosis and suppressing cell proliferation. However, the in vitro effect and detailed mechanism of Chinese medicinal herbs on metastasis of cancer cells was still unclear. Hence, a highly metastatic human lung cancer cell line, A549, was chosen to be treated with viola yedoensison extracts (VYE) and phyllanthus urinaria extracts (PUE) to investigate their potential for inhibiting cancer cell invasion. For a start, via modified Boyden chamber invasion assay, we found that VYE and PUE can suppress A549 cells invasion. And this two Chinese medicinal herbs did not have toxicity to A549 cells. Based on that tumor metastasis are accompanied with proteolytic degradation of the extracellular matrix and changed cell motility. Both of MMP-2 and u-PA activity were also inhibited by VYE and PUE via gelatin zymography and casein zymography assay. We also found that VYE and PUE can suppress the expression of proteinase and change proteinase inhibitor expression through RT-PCR and Western blotting. Furthermore, VYE could decrease the phosphorylation of p38, as well as suppressed the DNA binding activity and the nuclear level of nuclear factor-kappa B (NF-(NF-kappaB) and PUE could also suppress the phosphorylation of JNK, Akt, the binding activity and nuclear level of activator protein-1 (AP-1). In addition, A549-bearing BALB/c mice and LLC-bearing C57BL/6 mice were treated with VYE and PUE which tumor weight monitoring throughout the experiment, finally tumor weight, and lung metastases of animals have decreased. Finally, it was concluded that VYE and PUE may inhibit tumor metastasis via suppressing phosphorylation of MAPKs or Akt, the activity of transcription factors metastasis-related proteinase and affecting their inhibitor.

壹、中英文摘要……………………………………………1
貳、縮寫表…………………………………………………5
參、緒論……………………………………………………7
肆、研究動機………………………………………………21
伍、實驗材料與方法………………………………………22
陸、實驗結果………………………………………………36
柒、討論……………………………………………………46
捌、參考文獻………………………………………………52
玖、圖表與圖表說明………………………………………67
拾、附圖表…………………………………………………101

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