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研究生:王藝蓁
研究生(外文):Yi-Zhen Wang
論文名稱:樟芝子實體萃取物抑制人類胰臟癌細胞增生和誘導細胞凋亡之探討
論文名稱(外文):The extracts of Antrodia camphorate fruiting body inhibit cell proliferation and induce apoptosis on pancreatic cancer cells
指導教授:王志傑王志傑引用關係
指導教授(外文):"none"
學位類別:碩士
校院名稱:大仁科技大學
系所名稱: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:2011
畢業學年度:99
語文別:中文
論文頁數:114
中文關鍵詞:人類胰臟腺癌細胞細胞凋亡抗細胞增生樟芝
外文關鍵詞:Human pancreatic adenocarcinoma cellsapoptosisanti-proliferationAntrodia camphorata
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樟芝 (Antrodia camphorate) 為台灣特有的真菌類。在傳統中藥上,主要用於治療腹瀉、高血壓及保護肝臟等,已有研究指出樟芝具有增加免疫能力及抗癌等生理活性功能。本研究目的是利用體外細胞培養模式,研究不同溶劑之樟芝粗萃取物對人類胰臟腺癌細胞 (BxPC-3) 的生長抑制作用及誘導細胞凋亡之效應。本研究中將樟芝子實體粉末分別以水、乙醇及乙酸乙酯溶劑進行萃取,共製備三種粗萃取物。以 HPLC 測定萃取物之三萜類總面積量,分光光度計測定總多醣量以及使用螢光分析儀測定 1,3-β-D-glucan 之含量。總多醣含量分別是 399.11±0.01 mg/g、207.69±0.01 mg/g 及 140.64±0.03 mg/g;1,3-β-D-glucan 含量分別是 183.52±0.29 mg/g、84.01±0.36 mg/g 及 81.37±0.48 mg/g。其中三萜類總量以乙酸乙酯萃取物較高;總多醣及 1,3-β-D-glucan 之含量以水萃取物較多。以 MTT 分析法測定樟芝萃取物對 BxPC-3 細胞之毒殺作用,得知樟芝的水、乙醇及乙酸乙酯萃取物作用 BxPC-3 細胞 48 小時之百分之五十抑制濃度值 (IC50) 分別為 39.83±1.36 µg/mL、2.49±0.10 µg/mL 及 2.36±0.20 µg/mL。另外研究結果顯示,此三種樟芝萃取物均可促使人類胰臟腺癌細胞週期 Sub-G1 增加及促使細胞週期分布停滯在 G2/M 期。進一步利用 Annexin V-FITC/PI 細胞凋亡分析與 DNA 膠體電泳分析也驗證樟芝萃取物作用細胞 48 小時後確實會造成細胞凋亡。此外,我們也觀察到樟芝萃取物可造成細胞核內的染色質凝聚和核質碎裂,並且有凋亡小體產生之現象。而由細胞刮傷試驗結果顯示,三種樟芝萃取物都有抑制 BxPC-3 細胞遷移之能力,此抑制效應甚至比臨床用藥 Gemcitabine 更顯著且呈現時間性關係。最後,以西方墨點法進一步探討樟芝萃取物造成人類胰臟腺癌細胞凋亡機轉中與粒線體相關的訊息傳遞路徑蛋白之表現與活性。綜合本研究的結果,樟芝萃取物對 BxPC-3 細胞之增生,具有顯著的毒殺效力,且此毒殺效應可能經由粒線體相關訊息傳遞路徑之細胞凋亡機轉,本研究之發現期能提供樟芝做為臨床上抗胰臟腺癌之預防與治療。
Antrodia camphorate is a fungus native to Taiwan, and its fruiting body grows very slowly, thus becomes one of the most precious medicinal fungi. The fruiting body of A. camphorate is believed to be effective in diarrhea, abdominal pain, hypertension and anti-tumor. The goal of this study is to investigate the anti-proliferation effects of A. camphorate extracts (ACE) against BxPC-3 human pancreatic cancer cells in vitro, and the possibly involved apoptotic mechanism. Three extracts (W-E, E-E and EA-E) were obtained from the fruiting body of A. camphorata. The data showed that ACE contain triterpenoids, polysaccharides and 1,3-β-D-glucan, three major effective components which have been reported to have biological activities, and among them EA-E possessed a higher content of triterpenoids by HPLC analysis; whereas W-E and E-A possessed a higher content of polysaccharides by spectrophotometer analysis (W-E, 399.11±0.01 mg/g; E-E, 207.69±0.01 mg/g; EA-E, 140.64±0.03 mg/g) and 1,3-β-D-glucan by spectrofluor
-ometer analysis (W-E, 183.52±0.29 mg/g; E-E, 84.01±0.36 mg/g; EA-E, 81.37±0.48 mg/g). All extracts (W-E, E-E and EA-E) from ACE exhibited obvious cytotoxicity toward BxPC-3 cells for 48 h with IC50 values of 39.83±1.36 μg/mL, 2.49±0.10 μg/mL and 2.36±0.20 μg/mL, respectively. ACE induced a sub-G1 peak in flow cytometry histogram of treated cells indicating apoptosis is involved in this toxicity. Furthermore, in treating BxPC-3 cells for 48 h, the apoptotic effect of ACE was confirmed by annexin V-FITC/PI double staining and nuclear DNA fragmentation. Similarly, BxPC-3 cells treated with ACE were also found to reveal evident characteristics of apoptosis, which include characteristic chromatin condensation, cell blebbing, production of some tiny pellets, and formation of apoptotic bodies. Using wound healing assay, ACE significantly suppressed the migration ability of BxPC-3 cells in a time-dependent manner, and this inhibitory effect was even more significant than Gemcitabine, one clinical drug, which currently used for the treatment in pancreatic cancer therapy. Furthermore, Western blotting with treated cells demonstrated that ACE-induced apoptosis in BxPC-3 cells may occur through a mitochondria-dependent pathway while triggering an appropriate balance of bax/bcl-2, increasing levels of cytoplasmic cytochrome c, and activation of caspase-9, caspase-3 and PARP in. To conclude, we demonstrated that the fruit body extracts of A. camphorate exert the anti-proliferation and pro-apoptotic effects on BxPC-3 cells, which suggests that ACE seems to play a anticancer role in pancreatic cancer. It is possible that A. camphorate may serve as a potential naturally occurring compound for pancreatic cancer prevention and therapy.
中文摘要
英文摘要
縮寫表
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第一章 緒言
第二章 文獻回顧
第一節 樟芝介紹
第二節 胰臟癌
第三節 細胞凋亡
第四節 研究動機與目的
第三章 材料與方法
第一節 實驗材料與儀器設備
第二節 實驗藥品
第三節 樟芝萃取物之製備與成分分析
第四節 細胞培養
第五節 細胞存活率測定
第六節 細胞型態觀察
第七節 Hoechst 33258 細胞核染色法
第八節 細胞刮傷試驗
第九節 DNA 片段之分析
第十節 細胞週期分析
第十一節 Annexin V-FITC/PI 雙染分析
第十二節 西方墨點法分析 (Western blotting)
第十三節 統計分析
第四章 實驗結果
第一節 樟芝萃取物之製備及相關活性成份之測定
第二節 樟芝萃取物對 BxPC-3 細胞存活率之影響
第三節 樟芝萃取物對 BxPC-3 細胞型態觀察之情形
第四節 樟芝萃取物對 BxPC-3 細胞核之螢光染色觀察
第五節 樟芝萃取物對 BxPC-3 細胞遷移能力之情形
第六節 樟芝萃取物對 BxPC-3 細胞之 DNA 片斷化情形
第七節 樟芝萃取物對 BxPC-3 細胞週期之影響
第八節 樟芝萃取物處理 BxPC-3 細胞凋亡之影響
第九節 樟芝萃取物處理 BxPC-3 細胞之相關蛋白質表現
第五章 討論
第六章 結論與建議
第七章 參考文獻
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