臺灣博碩士論文加值系統

散腫潰堅湯 (San-Zhong-Kui-Jian-Tang , SZKJT)，是一個傳統的中藥方劑，已用在治療各種癌症的病人。據古書記載，其具有清熱解毒、破結潰堅之效能；亦即今日臨床之抗菌、抗腫瘤、鎮痛的作用；因此本研究首要在探討將散腫潰堅湯用在兩個乳癌細胞株 MCF-7及 MDA-MB-231 的抗癌效果。散腫潰堅湯藉由誘導癌細胞走向 G0/G1 phase arrest 及細胞凋亡 (Apoptosis)，而呈現出抑制細胞生長的作用。散腫潰堅湯可透過增加 p21/WAF1 表現，降低 cyclin D1、D2 的蛋白質量，而達到抑制細胞週期演進的效果；然而，由於散腫潰堅湯並不會增加 MCF-7 之 p53 的表現，因此都不是藉由 p53 的作用而來。使用散腫潰堅湯處理的癌細胞，會藉由改變 Bax/Bcl-2 的比例，而啟動粒線體細胞凋亡的路徑，使得 cytochrome c 釋放以及 caspase-9 活化；但並未作用在 Fas/Fas ligand 路徑及 caspase-8 的活化。在裸鼠實驗中更進一步的證明了散腫潰堅湯對癌細胞生長的抑制作用。因此我們的研究推測出對 p21/WAF1 的誘導作用以及活化粒線體細胞凋亡系統參與了散腫潰堅湯在人類乳癌細胞的抗增生活性。

San-Zhong-Kui-Jian-Tang (SZKJT; Japanese name: Sanshu-kaigen-to), a traditional Chinese medicine prescription, has been used for treating patients with various cancers. This study first investigates the anticancer effect of SZKJT in two human breast cancer cell lines, MCF-7 and MDA-MB-231. SZKJT exhibited effective cell growth inhibition by inducing cancer cells to undergo G0/G1 phase arrest and apoptosis. Blockade of cell cycle was associated with increased p21/WAF1 levels, and reduced amounts of cyclinD1, cyclinD2 in a p53-independent manner. SZKJT treatment triggered the mitochondrial apoptotic pathway indicated by changing Bax/Bcl-2 ratios, cytochrome c release and caspase-9 activation, but did not act on Fas/Fas ligand pathways and the activation of caspase-8. Further investigation revealed that SZKJT’s inhibition of cell growth effect was also evident in a nude mice model. Taken together, our study suggests that the induction of p21/WAF1 and activity of the mitochondrial apoptotic system may participate in the antiproliferative activity of SZKJY in human breast cancer cells.

中文摘要……………………………………………………………………….Ⅰ
英文摘要……………………………………………………………………….Ⅱ
本文目錄…….…………………………………………………………………Ⅲ
圖目錄………………………………………………………………………….Ⅵ
附圖目錄……………………………………………………………………….Ⅶ
縮寫表………………………………………………………………………….Ⅷ
第一章 緒論…………………………………………………………………….1
1-1 乳癌及其相關背景介紹………………………………………………..1
1-2 細胞週期與癌症………………………………………………………..3
1-3 細胞凋亡………………………………………………………………..6
1-4 散腫潰堅湯背景資料…………………………………………………11
第二章 材料與方法…………………………………………………………...12
2-1 材料……………………………………………………………………12
2-1-1 化學製劑…………………………………………………………12
2-1-2 原料植物…………………………………………………………12
2-2 方法………………………………………………………………..…..13
2-2-1 細胞培養 Cell Cultures………………………………………….13
2-2-2 細胞增生抑制分析 Cell Proliferation Assay (XTT)……………14
2-2-3 細胞週期分析 Cell Cycle Analysis……………………………..15
2-2-4 測定細胞凋亡 Apoptosis………………………………………..15
2-2-5 p53, p21/WAF1, Fas/APO-1 及 Fas Ligand (mFasL and sFasL) 之濃度分析……………………………………………………….16
2-2-6 Caspase-8 與 Caspase -9 活性分析……………………………..17
2-2-7 免疫墨點分析 Immunoblot Assay………………………………18
2-2-8 體內腫瘤異種移殖試驗 In Vivo Tumor Xenograft Study…….. 21
2-2-9 統計學分析………………………………………………………21
第三章 結果…………………………………………………………………...22
3-1 散腫潰堅湯對 MCF-7 及 MDA-MB-231 細胞株之細胞增生的影響…………………………………………………………………...…22
3-2 散腫潰堅湯對 MCF-7 及 MDA-MB-231 細胞株之細胞週期分佈的影響…………………………………………………………….……..22
3-3 散腫潰堅湯對 MCF-7 及 MDA-MB-231 細胞株誘導細胞凋亡. . …………………………………………………………………...22
3-4 散腫潰堅湯在與細胞週期相關分子上的影響………………………23
3-5 Fas/Fas Ligand 並不參與散腫潰堅湯相關的細胞凋亡……………..24
3-6 散腫潰堅湯啟動粒線體所主導的細胞凋亡路徑……………………24
3-7 散腫潰堅湯抑制裸鼠腫瘤生長………………………………………25
第四章 討論…………………………………………………………………...27
第五章 結論…………………………………………………………………...29
參考文獻……………………………………………………………………….30
圖表…………………………………………………………………………….41
附圖…………………………………………………………………………….50
圖目錄
圖1. 散腫潰堅湯對人類乳癌細胞株 MCF-7 和 MDA-MB-231 的增生抑制效果..…………………………………………………………………….41
圖2. 散腫潰堅湯對人類乳癌細胞株之細胞週期的影響..…………………..42
圖3 散腫潰堅湯誘導 MCF-7 及 MDA-MB231 之細胞凋亡…... ………..43
圖4. 散腫潰堅湯對細胞週期調控相關蛋白表現的影響..…………………..44
圖5. 散腫潰堅湯對死亡接受體凋亡性途徑中 Fas、mFas ligand 與 sFas ligand 濃度以及 caspase-8 活性的影響………………………………45
圖6. 腫潰堅湯透過粒線體路徑誘導細胞凋亡..………………………..........48
圖7. 潰堅湯抑制裸鼠的癌細胞生長……..…………………………………..49

附圖目錄
附圖1. ………….………………………………………………………………50
附圖2. ………………………….………………………………………...……51
附圖3. …………………………………………………………………………52
附圖4. …………………………………………………………………………53
附圖5. …………………………………………………………………………54

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