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研究生:李嘉偉
研究生(外文):Jia-Wei Li
論文名稱:牛樟芝子實體活性成分抑制前列腺癌之作用機轉
論文名稱(外文):The Anti-tumor mechanism of bioactive components isolated from Taiwanofungus camphoratus fruiting body on Human Prostate Cancer Cells
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:102
中文關鍵詞:細胞凋亡前列腺癌牛樟芝
外文關鍵詞:apoptosisprostate cancer cellsantrodia camphorata
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前列腺癌為男性最普遍的癌症之一,隨著年齡增加更為嚴重。在亞洲地區,近年來男性罹患前列腺癌的發生率及死亡率逐年增高。牛樟為台灣特有真菌,許多研究指出其具有抗癌效果。根據研究顯示牛樟芝子實體萃取物可抑制腫瘤停止生長及凋亡,但有關對於前列線癌細胞之影響的機制探討尚不明確。本研究使用牛樟芝子實體乙醇粗萃取物 ( ACCE ) 及牛樟芝管柱層析分離物 ( CFK4、CFK6 ) 進行實驗探討牛樟子實體萃取物對前列腺癌細胞 LNCaP 及 PC-3 的抑制效果,觀察是否能誘導前列腺癌細胞進行細胞凋亡。MTT assay 實驗中,以 ACCE、CFK4、CFK6 處理前列腺癌細胞 LNCaP 及 PC-3 經過 24 小時之後,細胞存活率會隨著萃取物的濃度增加而下降,前列腺癌細胞 LNCaP 及 PC-3 均對 CFK6 較為敏感,計算 CFK6 抑制前列腺癌細胞 LNCaP 及 PC-3 50% ( 50% inhibition concentration, IC50 ) 抑制濃度分別為 10.57 μg/ml 和 5.42 μg/ml。觀察細胞型態結果,隨著 CFK6 處理時間增加,前列腺癌細胞 LNCaP 及 PC-3 細胞型態不完整、細胞膜明顯皺縮、細胞膜上有 Phosphatidylserine ( PS ) 外翻情形。使用 PI 染色,發現前列腺癌細胞 LNCaP 及 PC-3 分別經由藥物處理過後 SubG1 比例增加,其次使用 DNA 電泳分析,對於前列腺癌細胞 LNCaP 及 PC-3 分別經由藥物處理過後,DNA 斷裂呈階梯狀片段,由以上兩種實驗顯示,牛樟芝子實體分離物 ( CFK6 ) 會促使前列腺癌細胞走向 apoptosis。最後在 Pan-caspase 活性測試及西方點墨法結果顯示,經不同濃度 CFK6 處理後前列腺癌細胞 LNCaP 及 PC-3,會活化 caspase-3、caspase-8、caspase-9、Fas-L 和 Bax 的蛋白表現量增加,而 Bcl-2 蛋白表現量減少,caspase-7 蛋白沒有明顯表現;在 p53 蛋白方面,前列腺癌細胞 LNCaP 隨著 CFK6 處理濃度增加,p53 蛋白明顯表現;前列腺癌細胞 PC-3 隨著 CFK6 處理濃度增加,p53 蛋白表現量較不明顯。由以上實驗結果證實,CFK6 對前列腺癌細胞 LNCaP 及 PC-3,主要凋亡路徑透過外部訊息傳遞路徑 Fas-L 蛋白表現,活化下游 caspase-8,活化的 caspase-8 會走向粒腺體傳遞路徑 Bax/Bcl-2,改變粒腺體膜電位釋放 cytochrome-c,來活化 caspase-9、caspase-3,而導致細胞凋亡。
The prostate cancer is one of the most common diseases for men and it becomes seriously with the age. In Asian area, the percentage and the death rate of the prostate cancer is increasing year by year for recent years. Taiwanofungus camphorates,also known as Antrodia camphorate (AC), is a kind of medical fungus only found in Taiwan. A lot of previous studies also confirmed that AC can be used for anticancer. According to studies that showed the extracts of AC fruiting body can make tumors be controlled or perished, but it’s effects for the prostate cancer cells were not fully understood. In this study, AC ethanol crude extracts (ACCE) and AC Column Chromatography extracts that named CFK4 and CFK6 were used to investigate that if AC fruiting body can make the prostate cancer cells, LNCaP and PC-3, be controlled. In this study, MTT assay showed that the cell survival rate decreased depending on the increase of dose, with ACCE、CFK4 and CFK6 were investigated on 24hr incubation. Both two tested prostate cancer were more sensitive to CFK6. This study showed that the IC50 of LNCaP and PC-3 were 10.57 μg/ml and 5.42 μg/ml. The results also showed that cellular morphological changes and cell shrinkage and the turn over of the Phosphatidylserine ( PS ) of Cell membrane when the disposal time of CFK6 was increased. By using PI staining, the data showed that the SubG1 of cell cycle was increased in prostate cancer cell, LNCaP and PC-3, treated by CFK6. DNA fragmentation analysis also confirmed that CFK6 could cause a apoptosis effect for both cell lines. According to the above experiment results, CFK6 made the two prostate cancer cell lines move toward apoptosis. Finally the assay about caspase activity and Western blot showed that LNCaP and PC-3 cells treated by increasing CFK6 could up regulate caspase-3, caspase-8, caspase-9 and the Bax ,but down regulate in Bcl-2 protein expression and caspase-7 was not changed. With the increasing concentration of CFK6, p53 protein in LNCaP and PC-3 also increased. Collectively, we showed CFK6 induced apoptosis through the regulation of FasL, p53, caspase-8, caspase-9, caspase-3, and the mitochondria pathway : Bax/Bcl-2 and cytochrome-c.
摘要.......................................................I
Abstract.................................................III
目錄.......................................................V
圖目錄..................................................VIII
表目錄.....................................................X
壹、前言....................................................1
貳、文獻回顧................................................3
一、前列腺及前列腺癌簡介………………………………………..3
(一)、前列線構造其特性…………………………………….3
(二)、前列線癌分佈及排行………………………………….4
二、前列腺癌分期及診斷……………………...…………………...5
(一) 前列線癌分期.…………………………………………5
(二) 前列腺癌診斷.…………………………………………5
三、前列腺癌治療方式……………………………………………..7
(一) 早期前列腺癌………………………………………….7
(二) 中期前列腺癌………………………………………….7
(三) 後期前列腺癌………………………………………….8
四、牛樟芝簡介……………………………………………………..8
(一) 牛樟芝命名…………………………………………….8
(二) 牛樟芝形態…………………………………………...10
五、牛樟芝成分分析……………………………………………....11
(一) 一般成分……………………………………………...11
(二) 生理活性成分………………………………………...12
六、牛樟芝生理活性功能………………………………………....18
(一) 抗氧化功效…………………………………………...18
(二) 癌細胞毒殺功效……………………………………...19
(三) 肝臟防護功能………………………………………...21
(四) 增進免疫功效………………………………………...22
(五) 抗病毒能力…………………………………………...23
(六) 抗發炎能力…………………………………………...23
(七) 降血糖能力…………………………………………...24
七、牛樟芝的安全性……………………………………………....25
(一) 急性毒性試驗………………………………………...25
(二) 亞急性毒性試驗……………………………………...25
(三) 微生物毒性試驗……………………………………...25
八、細胞凋亡作用形態特徵………………………………………26
(一) 細胞凋亡形態學……………………………………...26
(二) 細胞凋亡生化特徵…………………………………...27
九、細胞凋亡的訊息傳遞路徑……………………………………28
十、p53 抑癌基因…………………………………………………30
参、研究目的……………………………………………………………33
肆、實驗架構……………………………………………………………35
伍、實驗材料與方法……………………………………………………37
一、牛樟芝子實體萃取……………………………………………37
二、利用管柱層析法分離萃取物…………………………………37
三、細胞培養與測試………………………………………………38
(一) 細胞培養……………………………………...............38
(二) 細胞毒性測試…………………………………...........39
四、細胞凋亡現象測試……………………………………………40
(一) 前列腺癌細胞形態觀測……………………………...40
(二) 細胞週期分析…………………………………….......41
(三) 利用DNA 片段電泳分析……………………………42
(四) 利用Pan-caspase 活性套組檢測…………………….43
五、利用西方墨點法分析凋亡路徑………………………………43
陸、結果…………………………………………………………………47
一、牛樟芝子實體乙醇萃取物成分分離…………………………47
二、細胞存活率測試………………………………………………47
三、前列腺癌細胞型態影響………………………………………49
四、細胞週期凋亡現象影響………………………………………50
五、DNA片段化現象測試………………………………………...51
六、pan caspase 活性套組測試…………………………………...52
七、細胞凋亡路徑測試……………………………………………52
柒、討論…………………………………………………………………81
捌、結論…………………………………………………………………83
玖、未來展望……………………………………………………………85
參考文獻………………………………………………………………..87
附錄一 乙醇毒性測試………………………………………………..97
附錄二 CFK4、CFK6 對纖維母細胞毒性測試……………………...98
附錄三 反應試劑配方………………………………………………..99
附錄四 p53凋亡機制調控圖………………………………………..101
附錄五 細胞凋亡路徑圖……………………………………………102
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