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研究生:曾乙召
研究生(外文):Yi-Chao Tseng
論文名稱:桑黃固態發酵產物之生物活性探討
論文名稱(外文):The bioactivity study of mycelium from solid-state fermentation of Phellinus linteusThe bioactivity study of mycelium from solid-state fermentation of Phellinus linteusThe bioactivity study of mycelium from solid-state fermentation of Phellinus linteusThe bioactivity study of mycelium from solid-state fermentation of Phellinus linteus
指導教授:陳健祺
學位類別:碩士
校院名稱:南台科技大學
系所名稱:生物科技系
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:102
畢業學年度:101
語文別:中文
論文頁數:101
中文關鍵詞:桑黃抗腫瘤轉移試驗BaxBcl-2caspase-9caspase-3抗氧化
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  • 被引用被引用:1
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中文摘要
在東方社會裡,桑黃 (Phellinus linteus)是一種被廣泛使用的藥用菇類,在傳統民間中醫治療上,桑黃具有止血、治腹痛、利尿、健胃、止瀉等治療功效,過去研究指出桑黃具有免疫調節、抗腫瘤、抗氧化等功能。但是,因桑黃子實體生長緩慢且不易取得,故本研究想透過培養基的組成改變及培養條件的不同而得到不同的發酵產物,並進而探討相關的生物活性。
結果顯示,固態發酵桑黃菌絲體水萃物能夠有效抑制乳癌細胞 (MDA-MB-231、MCF-7)的生長,並再進一步使用流式細胞儀去探討桑黃固態發酵水萃物作用在乳癌細胞上,細胞週期的相關機制及變化。隨著水萃物濃度越高,則使乳癌細胞停滯在細胞週期中的G2/M期表現越為明顯。接著利用RT-PCR和Western blot等方法探討G2/M期的相關因子,發現桑黃在細胞週期素cyclin A、cyclin B1、cdc2等相關因子及促進細胞週期停滯因子p21的mRNA及蛋白質的表現量卻沒有明顯差異,但是細胞存活試驗能夠有效抑制乳癌細胞的增生,因此接著探討粒線體凋亡路徑等相關蛋白質,發現桑黃菌絲體水萃物在乳癌細胞(MDA-MB-231、MCF-7)上能夠調控Bax、caspase-9、caspase-3等蛋白質的表現量上升及抑制抗凋亡蛋白Bcl-2的表現。

在抑制轉移試驗,使用固態發酵桑黃菌絲體水萃物處理人類纖維瘤細胞 (HT-1080),明顯抑制基質金屬蛋白酶 (MMP-9)的分泌。並在RT-PCR實驗中使用桑黃固態發酵水萃物作用在細胞株上,同樣也是抑制MMP-9及MMP-2 mRNA的表現。在免疫調節試驗中,使用固態發酵桑黃菌絲體水萃物刺激巨噬細胞 (Raw 264.7)分泌細胞激素,進而間接抑制乳癌細胞的生長,此外也使用流式細胞儀偵測中國倉鼠卵巢細胞CHO-K1細胞氧化程度,結果顯示桑黃菌絲體水萃物可抑制H2O2對CHO-K1細胞內產生的氧化傷害。
綜合以上結果顯示,固態發酵桑黃菌絲體水萃物對於乳癌細胞株有效的抑制生長,並且使細胞停滯在G2/M期、促使細胞走向粒線體凋亡路徑、抑制轉移因子MMP-9、MMP-2的表現、間接提升免疫功效及抗氧化等生物活性。
關鍵字:桑黃(Phellinus linteus)、抗腫瘤、轉移試驗、cyclin A、cyclin B1、cdc2、p21、Bax、Bcl-2、caspase-9、caspase-3、MMP-9、MMP-2、抗氧化
Abstract
Sangwhang (Phellinus linteus) is a widely used medicinal mushroom in oriental society. The bioactivities of P. linteus such as immunomodulatory, anti-tumor and anti-oxidation have been reported in previous studies. However, the fruit body of P. linteus grows slowly and is difficult to obtain. In order to explore the bioactivities of P. linteus, the products of solid-state fermentation from P. linteus were used in this study. The results showed that the water extract of mycelium from solid-state fermentation of P. linteus significantly inhibited the growth of breast cancer cells (MCF-7 and MDA-MB-231) evaluated by MTT assay. The growth-inhibitory effect on breast cancer cells was due to arrest MCF-7 or MDA-MB-231 cells in G2/M phase evaluated by flow cytometer. Furthermore, RT-PCR assay and Western blot analysis were used to illustrate the related factors of G2/M phase. The results showed that water extract of mycelium from solid-state fermentation of P. linteus had no significant effect on cyclin A, cyclin B1 and cdc2, and p21 expression of mRNA and protein. According to MTT data, however, these samples were able to significantly inhibit the growth of breast cancer cells. Thus, breast cancer cells (MDA-MB-231, MCF-7) treated with water extract of mycelium from P. linteus would undergo to find out the mitochondrial apoptotic pathway by Western Analysis. Moreover, the related proteins, such as Bax, caspase-9, caspase-3 were examined. The results showed that the expressions of Bax, caspase-9, and caspase-3 were increased, and the anti-apoptotic protein Bcl-2 was inhibited. In the gelatin zymography assay, the results showed that the water extract of mycelium from solid-state fermentation of P. linteus effectively inhibited the matrix metalloproteinase-9 secretion on HT-1080 cells. The mRNA expressions of MMP-9 and MMP-2 were also suppressed while cells were treated with water extract of mycelium from solid-state fermentation of P. linteus. In immunomodulatory study, the results showed that the water extracts of mycelium from solid-state fermentation of P. linteus stimulated the cytokine secretion of Raw 264.7 cells to inhibit the growth of breast cancer cells. Furthermore, the antioxidant activity was measured by FASCAN, the data showed that the water extract of mycelium from solid-state fermentation of P. linteus significantly decreased ROS level. According to our study, the results demonstrated that water extract of mycelium from solid-state fermentation of P. linteus can significantly inhibit the cellular growth, arrest cell at the G2/M phase, and inhibit MMP-9 expression and enhance the ability of antioxidation.
Keyword:Phellinus linteus、anti-tumor、gelatin zymography assay、cyclin A、cyclin B1、cdc2、p21、Bax、Bcl-2、caspase-9、caspase-3、MMP-9、MMP-2、anti-oxidation
目錄
中文摘要 i
英文摘要 iii
第一章 文獻回顧 1
一、桑黃菌(Phellinus linteus)
1.1桑黃的分類及特徵 1
1.2桑黃之生物學特性 1
二、桑黃菌之化學組成 2
三、癌症 3
3.1乳癌的介紹 3
3.2乳癌的治療 4
四、癌細胞的細胞週期 4
4.1細胞週期(Cell Cycle) 5
4.2 G2/M checkpoint 5
五、癌細胞的轉移 6
六、免疫調節 7
七、自由基 7
第二章 研究動機 9
第三章 實驗架構 10
第四章 材料方法 11
一、實驗儀器與藥品 11
1.1桑黃菌培養基 11
1.2實驗細胞株 11
1.3實驗藥品 11
1.3.1購自SIGMA(USA) 11
1.3.2購自MERCK(USA) 12
1.3.3購自AMERSCO(USA) 12
1.3.4購自Bio-Rad(USA) 12
1.3.5購自VIOGENE 12
1.3.6購自GIBCO(USA) 12
1.3.7購自Amersham (UK) 12
1.3.8購自GE Healthcare 12
二、 樣品製備 13
2.1 固態培養桑黃菌絲體 13
2.2 樣品萃取製備 13
三、 實驗方法 13
3.1細胞存活率測試( MTT assay ) 13
3.2細胞週期測定 14
3.3反轉錄聚合酵素鏈鎖反應
( RT-PCR,reverse transcription polymerase chain reaction) 15
3.3.1 抽取mRNA 15
3.3.2反轉錄聚合酶連鎖反應(RT-PCR) 15
3.3.3電泳分析法cDNA分析片段(Agarose gel electrophoresis) 16
3.4西方墨點法(Western blot) 17
3.4.1原理 17
3.4.2實驗步驟 17
3.5 Matrix Metalloproteinases的收集與定量 19
3.5.1 Conditioned medium與細胞溶解液(cell lysate)的收集 19
3.5.2 蛋白質含量的測定 19
3.6電泳分析法(Gelatin Zymography Assay) 20
3.6.1 SDS-PAGE Electrophoresis 與染色步驟 20
3.7抗氧化試驗 20
3.7.1 細胞氧化程度偵測原理 20
3.7.2 實驗步驟 21
3.7.3 抗氧化能力原理 21
3.7.4 抗氧化能力實驗步驟 22
3.8提升免疫能力測試 22
3.8.1 抑制癌細胞株生長試驗 22
3.8.2 提升免疫能力 23
3.9統計分析 23
第五章 實驗結果 24
一、 抑癌試驗 24
1.1 抑制腫瘤細胞增生試驗 24
1.2 固態發酵桑黃菌絲體水萃物對乳癌細胞週期影響 25
1.3固態發酵桑黃菌絲體水萃物對乳癌細胞cyclin A、cyclin B1、cdc2
和p21mRNA與蛋白質的表現之影響 27
1.4固態發酵桑黃菌絲體水萃物對乳癌細胞細胞凋亡因子蛋白質之影響 28
二、轉移酵素試驗 29
2.1 固態發酵桑黃菌絲體水萃物對人類纖維母細胞(HT-1080) MMP-9、
MMP-2 mRNA表現的影響 29
2.2 固態發酵桑黃菌絲體水萃物對人類纖維母細胞(HT-1080) MMP-9、
MMP-2 蛋白質表現的影響 29
三、固態發酵桑黃菌絲體水萃物抗氧化能力測定 30
3.1細胞抗氧化測定 30
3.2固態發酵桑黃菌絲體水萃物抗氧化能力 30
四、提升免疫能力測試 31
4.1間接免疫測試 31
4.2提升免疫能力 31
第六章 討論 33
ㄧ、抑制腫瘤細胞存活試驗 33
二、細胞週期變化試驗與週期因子探討 34
三、細胞凋亡因子探討 35
四、 MMPs 活性試驗 36
五、固態發酵桑黃菌絲體水萃物抗氧化測定 37
六、間接免疫與免疫能力活性試驗 38
第七章 結論 40
第八章 未來計畫 41
第九章 參考文獻 42
第九章 參考文獻
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