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研究生:吳佳燕
研究生(外文):Chia-Yen Wu
論文名稱:牛樟芝子實體萃取物對於人類大腸癌細胞株 caco-2之細胞凋亡機制探討
論文名稱(外文):The apoptotic mechanism of Taiwanofungus camphoratus fruiting bodies’ extracts on human colon cancer caco-2 cell line
指導教授:吳宗正吳宗正引用關係
指導教授(外文):Tzong-Zeng Wu
學位類別:碩士
校院名稱:國立東華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
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:105
中文關鍵詞:牛樟芝大腸癌大腸癌細胞株
外文關鍵詞:Taiwanofungus camphoratuscolon cancercaco-2
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本實驗中利用矽膠管柱層析法,將牛樟芝子實體粗萃物依序由低極性至高極性逐步萃取,依極性遞減方式,分成FK1至FK6等六個劃分群,將各劃分群以不同劑量處理 caco-2 人類大腸癌細胞株,並利用細胞存活率測試篩選出最有效劃分為FK6。結果顯示FK6之IC50劑量為20 μg / mL。Caco-2細胞株經FK6以 IC50劑量處理後,細胞週期中subG1期於24小時增加7.3%、48小時增加8.9%。接著以annexinV - FITC&;PI雙染,結果顯示隨著加入FK6 濃度或時間增加,偵測到活著的細胞數量下降,壞死及凋亡的細胞數量上升。最後利用Western blot偵測凋亡蛋白表現,證實 caco-2 細胞株受到刺激後,在extrinsic apoptosis部分,FasL表現量增加,活化死亡受體Fas receptor;接著活化細胞質內caspase 8,促使Bid活化成tBid,造成粒線體中Bax表現量上升與Bcl-2表現量下降,而誘導粒線體中cytochrome c 釋出,充分顯示出intrinsic apoptosis中粒腺體訊息傳遞路徑,cytochrome c 由粒腺體釋出後,會活化caspase 9,接著活化下游caspase 3,再進一步活化caspase 7並抑制PARP 表現,使細胞喪失DNA修復能力,促使細胞走向凋亡。本論文證實牛樟芝子實體萃取物中的FK6 成分,能夠抑制 caco-2 癌細胞的生長,並誘導細胞引發Fas / FasL 下游一連串凋亡訊息產生,細胞因而進行凋亡反應。基於本論文所顯示的成果,牛樟芝子實體中 FK6 成分,應有機會進一步成發展成治療大腸癌之有效藥物。
In this study, the crude extract of Taiwanofungus camphoratus fruiting body was sequentially separated into six fractions, FK1 to FK6, according to the polarity of components by using column chromatography process. These fractions were used to treat human colorectal cancer cell line caco-2 with different doses. The result showed that the IC50 of FK6 was 20 μg / mL. By using PI staining, the data showed of subG1of cell cycle was increased 7.3% after treating 20 μg / mL of FK6 for 24 hr., and increased 8.9% after treating 48hr. in caco-2 cancer cell. Subsequently, the results of annexinV - FITC & PI double staining showed that the number of living cells was decreased, and the number of necrosis and apoptotic cells was increased along with the increasing dose of FK6 and treating time. Finally, the results of Western blot showed that FK6 induced FasL expression and interacted with Fas receptor to activate caspase 8 in extrinsic apoptosis pathway. In intrinsic apoptosis pathway, caspase 8 activated Bid into tBid, it caused the increase of Bax expression and decrease of Bcl-2 expression inmitochondria, afterward induced cytochrome c released from mitochondria to cytoplasm. The above results fully demonstrated the intrinsic apoptosis pathway in mitochondria. While cytochrome c released from mitochondria, it activated caspase 9, followed by activation of downstream caspase 3, caspase 7 and inhibited PARP performance. Therefore cell lossed DNA repaired capacity, and promoted cells to apoptosis.
This paper indicated that FK6 fraction of Taiwanofungus camphoratus extract induced colorectal cancer cell line caco-2 apoptosis through Fas / FasL signal pathway activation which inhibited DNA repair capability. Based on the results showed in this paper, the FK6 ingredient of Taiwanofungus camphoratus fruiting bodies should has the opportunity to further develop the effective medicine for treatment of colon cancer.
中文摘要 I
Abstract III
目錄 IV
表目錄 VIII
圖目錄 IX
壹、前言 1
貳、文獻回顧 3
一、牛樟芝簡介 3
二、牛樟芝成分分析 6
三、牛樟芝菌絲體與子實體成分比較 11
四、牛樟芝子實體抑癌功效 13
五、大腸直腸癌簡介 15
參、研究動機及目的 23
研究動機 23
研究目的 24
研究架構 25
肆、實驗材料與儀器 27
一、實驗材料 27
二、實驗耗材 28
三、實驗儀器 29
伍、實驗方法 31
一、牛樟芝子實體萃取 31
二、牛樟芝菌絲體萃取 33
三、人類大腸癌細胞株caco-2培養 33
四、細胞存活率測試 33
五、細胞凋亡測試 34
(一)、顯微鏡觀察螢光染色細胞 34
(二)、細胞週期分析 34
(三)、細胞 annexinV&PI雙染分析 35
(四)、DNA 片斷化 36
(五)、Pan – caspase 分析 36
六、西方點墨法 37
蛋白質萃取 38
蛋白質定量 38
蛋白質電泳極轉漬 38
蛋白質免疫呈色 38
七、統計分析 39
陸、實驗結果 41
一、索氏萃取子實體 41
二、子實體粗萃物管柱層析 41
三、樟芝子實體和菌絲體粗萃取物對 caco-2細胞株存活率比較
..41
四、牛樟芝子實體酒精粗萃物對 caco-2 細胞週期分析 42
五、子實體管柱分離萃取物 (FK1~FK6) 對於 caco-2 細胞株存活率之影響 42
六、FK6 對人類正常纖維母細胞之影響 43
七、FK6處理 caco-2細胞株後,細胞週期分析 43
八、caco-2 處理 FK6後,加入 annexinV&PI染劑顯微鏡觀察圖 44
九、caco-2處理 FK6 後,加入 annexinV&PI 染劑,流式細胞儀分析 44
十、DNA片斷化實驗 45
十一、Pan – caspase 蛋白活性實驗 46
十二、西方點墨法 47
柒、討論 53
捌、結論 93
玖、未來研究方向 95
拾、參考文獻 97
附錄 104
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