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研究生:賴鈺菁
研究生(外文):Yu-Ching Lai
論文名稱:樟芝發酵液之抗發炎及其誘導癌細胞凋亡機制之探討
論文名稱(外文):Anti-inflammatory effect and induction of apoptosis in cancer cells by Antrodia Camphorata
指導教授:楊新玲楊新玲引用關係許游章
指導教授(外文):Hsin-Ling YangYou-Cheng Hseu
學位類別:碩士
校院名稱:中國醫藥大學
系所名稱:營養研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:148
中文關鍵詞:樟芝發酵液脂多醣誘導型一氧化氮合成酶環氧化酶-2一氧化氮人類急性骨髓血癌細胞人類雌激素依賴型乳癌細胞細胞凋亡
外文關鍵詞:Fermented culture broth of A. camphorata ( FCBA)Lipopolysaccharide (LPS)Inducible nitric oxide synthases (iNOS)Cyclooxygenase-2COX-2Nitric oxide (NO)Human premyelocytic leukemia cells (HL60 cells)Human Estrogen-dependent Breast Cancer Cells (MCF-7)Apoptosis
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  • 被引用被引用:18
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樟芝 (學名Antrodia camphorata) 是生長在牛樟樹上的一種菇類,而牛樟樹為台灣特有。相傳樟芝具有解毒、抗腫瘤、抗過敏及降血糖等功能。本研究之一是利用樟芝發酵液(Fermented culture broth of A. camphorata, FCBA)為材料,探討其對脂多醣(Lipopolysaccharide, LPS)所誘發巨噬細胞 (RAW 264.7)發炎反應之影響。研究發現,FCBA可抑制LPS (1μg/ml)誘發巨噬細胞所產生之一氧化氮(Nitric oxide) 含量。在蛋白質分析結果顯示其亦可有效抑制COX-2(cyclooxygenase-2)、iNOS (inducible nitric oxide synthase)的表現及IκB-α的降解作用。
另一研究是以FCBA作用於人類急性骨髓血癌細胞(Human Premyelocytic Leukemia Cells,HL-60)及人類雌激素依賴型乳癌細胞(Human Estrogen-dependent Breast Cancer Cells, MCF-7)中,探討其抑制HL-60、MCF-7的細胞存活率及生長的情形及可能作用機制。利用電子顯微鏡及DNA電泳法,發現FCBA可以誘導HL-60、MCF-7細胞凋亡,特徵包括細胞皺縮(cell shrinkage)、存活率(viability)下降和DNA斷裂(DNA fragmentation);且隨FCBA處理濃度及作用時間的增加而增加。利用化學冷光法(chemiluminescence assay)去偵測FCBA作用於MCF-7上的活性氧化物(reactive oxygen species, ROS)變化,結果發現MCF-7之ROS量隨FCBA處理濃度的增加而增加。利用西方點墨法(Western blotting)偵測FCBA對HL-60、MCF-7細胞的Bcl-2、Bax和Cytochrome c表現,結果發現(1)HL-60的Bcl-2蛋白質受FCBA抑制而Bax蛋白質量增加,而MCF-7的Bax蛋白質有增加,但Bcl-2蛋白質並無顯著變化;(2)兩株癌細胞的Cytochrome c蛋白質合成量皆顯著增加。此外,(3)PARP皆可被切成兩片段(116和89 kDa)及(4)Caspase 3皆被活化。因此,樟芝會誘導癌細胞凋亡,推測是因ROS的產生,導致bax增加(及bcl-2減少),使cytochrome c被釋放而活化Caspase 3,進而使PARP被切斷的路徑所致。
總結以上結果,樟芝發酵液(Fermented culture broth of A. camphorata,FCBA)具有抗發炎、抗腫瘤之功效。
Antrodia camphorata(A. camphorata)is well known in Taiwan as a traditional Chinese medicine, and it has been shown to exhibit antioxidant and antitumor effects. In this study, we investigated anti-inflammatory me- chanisms effect of fermented culture broth of A. camphorata(FCBA).In
previous studies, we have shown that FCBA(25-100 ug/ml) not only succ- essfully inhibited lipopolysaccharide(LPS, 1 ug/ml)-induced inflammatory responses, but also decreased nitrite production in macrophage cells(RAW 264.7). Furthermore, FCBA inhibited iNOS(inducible nitric oxide syntha- se), COX-2(cyclooxygenase-2)protein expression and IκB-α degradation by using Western blot analysis. These results suggested that FCBA have anti-inflammatory effect via suppressing the iNOS and COX-2 expression through inhibition of IκB-α degradation.
In this study, the ability of A. camphorata to induce apoptosis was stu- died in cultured HL-60(human premyelocytic leukemia cells) and MCF-7(Human Estrogen-dependent Breast Cancer Cells.Treatment of the HL-60 and MCF-7 cells with a variety of concentrations of the ferme- nted culture broth of A. camphorata (25-150 g/ml) resulted in dose- and time-depend- ent sequences of events marked by apoptosis, as shown by loss of cell via- bility, chromatin condensation, and internucleosomal DNA fragmentation. In chemiluminescence assay, FCBA in MCF-7 can caused an increase of reactive oxygen specieses(ROS). Further more, apoptosis in the HL-60 and MCF-7 cells were measurement by western blotting.(1)in the HL-60, the increase of Bax protein and reduction of Bcl-2 protein; in the MCF-7, the increase of Bax protein but Bcl-2 protein is no significantly different.(2)they can release of cytochrome c and can (3)activation of specific proteoly- tic cleavage of poly (ADP-ribose) and caspase 3. The data sug- gested that A. camphorata exerts antiproliferative action and growth inhibition on HL-60 cells and MCF-7 cells through apoptosis induction, and that it may have anticancer properties valuable for application in drug products.
第一部分 樟芝發酵液之抗發炎影響
壹、 摘要……………………………………… … ……… 10-12
貳、 前言及文獻背景…………………………………….. 13-30
參、 實驗材料…………………………………………….. 31-33
肆、 實驗方法……………………………………………...34-44
伍、 實驗結果及圖表……………………………………...45-52
陸、 討論…………………………………………………...53-56
第二部分 樟芝發酵液誘導癌細胞凋亡機制探討
壹、 摘要……………………………………… … ……… 58-60
貳、 前言及文獻背景…………………………………….. 61-80
參、 實驗材料……………………………………………...81-84
肆、 實驗方法……………………………………………...85-100
伍、 實驗結果及圖表…………………………………….101-125
陸、 討論………………………………………………….126-129
總結………………………………………………………….130-131
參考文獻…………………………………………………… 132-148
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