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研究生:王宏聖
研究生(外文):Wang, Hung-Sheng
論文名稱:桑黃液態發酵產物之生物活性探討
論文名稱(外文):The bioactivity study of fermented liquid from Phellinus igniarius
指導教授:陳健祺
指導教授(外文):Chen, Jian-Chyi
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:100
畢業學年度:99
語文別:中文
論文頁數:85
中文關鍵詞:桑黃Phellinus igniarius抗腫瘤免疫調節抗氧化抗轉移
外文關鍵詞:Phellinus igniariusantitumorimmunomodulatoryantioxidantp21cyclin Acyclin Bcdc2MMP-2MMP-9.
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桑黃是一種珍貴的藥用菇類被廣泛地運用在東方社會,根據先前的研究指出桑黃具有抗腫瘤、免疫調節、抗氧化、抗發炎、等功效,因此本研究以不同碳源液態發酵桑黃產物(菌絲體水萃物、發酵液、純化多醣)進行生物活性探討。
結果顯示,液態發酵桑黃菌絲體水萃取物能夠顯著抑制乳癌細胞(MCF-7、MDA-MB-231)的生長,進一步利用流式細胞儀在細胞週期上的探討得知其機制主要是抑制乳癌細胞的細胞週期,使停滯在G2/M期,透過RT-PCR及Western blot方式發現桑黃具有促進細胞週期停滯因子p21的表達及抑制cyclin A、cyclin B cdc2等細胞週期因子,導致細胞週期停滯。
在抑制轉移試驗中發現桑黃菌絲體水萃物處理HT-1080顯著抑制金屬蛋白酶MMP-9的分泌,此外在RT-PCR試驗中也發現經由桑黃菌絲體水萃處理後細胞MMP-2及MMP-9 mRNA表現也受到抑制。在免疫調節試驗中藉由桑黃發酵液及多醣刺激Raw264.7分泌細胞激素抑制乳癌細胞生長,且透過RT-PCR證實桑黃多醣及發酵液能提升Raw264.7分泌細胞激素TNF-α、IL-1與IL-6達到提升免疫功效。在DCFH-DA抗氧化能力試驗中藉由流式細胞儀發現桑黃菌絲體水萃取物具有抗氧化活性,能夠減少CHO-K1細胞內ROS含量。
綜合以上結果證實了桑黃確實有抗腫瘤、抑制轉移以及抗氧化之生物活性,我們實驗結果顯示桑黃是值得進一步深入研究與開發。
Sangwhang (Phellinus igniarius) is a precious medical mushroom and was widely used in oriental society. It have been reported to possess important biological functions including anti-tumor, immunomodulatory, anti-oxidant, anti-inflammatory etc. The aim of this study was to investigate the biological effects of cultural broth and mycelium of P. igniarius.
The results have shown that the fermented liquid and water extract of mycelium from P. igniarius can significantly inhibit the growth of breast carcinoma cells according to MTT-assay. The growth-inhibition effect of P. igniarius on breast carcinoma cells was due to arrest breast carcinoma cells in G2/M phase by flow cytometer. Moreover, the fermented liquid and water extract of mycelium from P. igniarius could also up-regulate the p21 mRNA expression and down-regulate the protein level of cyclin A, cyclin B and cdc2, leading to induction of G2/M cell-cycle arrest in MCF-7 and MDA-MB-231 cells.
According to gelain zymorgraphy analysis, the result had showed that the secretion of MMP-9 protein was obviously suppressed while HT-1080 cells were treated by fermented liquid and water extract of mycelium from P. igniarius. In addition, the mRNA expression of MMP-2 and MMP-9 was also suppressed while cells treated by water extract from the mycelium of P. igniarius.
The immunomodulatory effect of P. igniarius was also investigated during this study. The results showed that the conditional medium from Raw 264.7 cells treated with fermented liquid and polysaccharide from P. igniarius had the significantly growth-inhibitory effect in MDA-MB-231 and MCF-7 cells. Furthermore, the mRNA expression of cytokines released from Raw 264.7 cells was analyzed by RT-PCR assay. The results demonstrated that fermented liquid and polysaccharide from P. igniarius had immunomodulatory effect on Raw 264.7 cells by the increment of the IL-1、IL-6 and TNF- mRNA expression. Finally, the antioxidant activity was measured by FASCAN, and thedata showed that the water extract of mycelium from P. igniarius can significantly reduce ROS which generated from H2O2. .
Our findings suggested P. igniarius indeed possess biological activities, such as antitumor, antimetastatsis and antioxidant activities. Therfore, it is worthy to do more studies to elucidate the functional activities of P. igniarius.
中文摘要 i
英文摘要 iii
第一章 文獻回顧
一、桑黃菌(Phellinus igniarius)簡介
1.1桑黃的分類及特徵 1
1.2桑黃之生物學特性 1
1.3桑黃之功效 2
二、桑黃菌之化學組成 2
2.1桑黃多醣之組成 3
三、桑黃之生物活性與藥理療效 5
3.1抗腫瘤活性 5
3.2提升免疫 6
3.3抗發炎反應 6
3.4抗氧化 7
3.5抗血管增生 8
3.6降血糖、降血脂 8
四、癌症 8
4.1乳癌的介紹 9
4.2乳癌的治療 9
五、癌細胞的細胞週期與轉移 10
5.1細胞週期 10
5.2G2/M檢查點激酶介紹 11
5.3轉移 13
第二章 研究動機 14
第三章 實驗架構 15
第四章 材料方法 16
一、實驗儀器與藥品 16
1.1桑黃菌培養基 16
1.2實驗細胞株 16
1.3實驗藥品 16
1.3.1 購自SIGMA (USA) 16
1.3.2購自波仕特公司 17
1.3.3購自德國MERCK 17
1.3.4 購自美國Amersham 17
1.3.5 購自Bio-Rad 17
1.3.6 購自VIOGENE 17
1.3.7 抗體使用一覽表 18
1.3.8購自Gibco 18
1.3.9引子( Primer ) 18
1.4實驗儀器 19
二、實驗方法 21
2.1 樣品製備 21
2.1.1液態培養桑黃菌絲體與發酵液處理 21
2.1.2胞外多醣之純化 21
2.1.2.1去蛋白 21
2.1.2.2酒精沉澱 22
2.2 細胞存活率測定(MTT assay) 22
2.2.1細胞存活率之測定原理 22
2.2.1.1實驗步驟 22
2.3 Matrix Metalloproteinases的收集與定量 23
2.3.1實驗步驟 23
2.3.1.1 Conditioned medium與細胞溶解液(cell lysate)的收 24
2.3.1.2蛋白質含量的測定 24
2.4電泳分析法(Gelatin Zymography Assay) 24
2.4.1 SDS-PAGE Electrophoresis 與染色步驟 25
2.5 西方墨點法(Western blot) 25
2.5.1原理 25
2.5.2實驗步驟 26
2.6細胞週期測定透過流氏細胞儀(Flow Cytometer,FACS) 28
 2.6.1原理 28
2.6.2實驗步驟 28
2.7反轉錄-聚合酵素連鎖反應(reverse transcription polymerase chain reaction, RT-PCR)
2.7.1原理 29
2.7.2 實驗步驟 29
2.7.2.1mRNA萃取 29
2.7.2.2反轉錄聚合酶連鎖反應(RT-PCR) 30
2.7.2.3電泳分析法cDNA分析片段(Agarose gel electrophoresis) 31
2.8抗自由基試驗 31
2.8.1細胞氧化程度偵測原理 31
2.8.2實驗步驟 31
2.9提昇免疫能力測試 32
2.9.1 步驟 32
2.9.2 抑制癌細胞株生長試驗 32
2.10統計分析 33
第五章 實驗結果 34
一、 抑癌試驗 34
1.1 抗癌腫瘤細胞增生試驗 34
1.2 液態發酵桑黃菌絲體對乳癌細胞週期影響 35
1.3 桑黃菌絲體水萃物(F-W或M-W)對乳癌細胞p21、cyclin A、 cyclin B mRNA表現之影響 37
1.4 桑黃菌絲體水萃物(F-W或M-W)對乳癌細胞cyclin A、 cyclinB、cdc2 protein表現之影響 38
1.5 桑黃F-W、M-W菌絲體水萃物對乳癌細胞MDA-MB-231貼附之影響 39
二、轉移酵素試驗 39
2.1 桑黃F-W、M-W菌絲體水萃物對細胞MMP-2、9 mRNA表現之影響 40
三、提升免疫能力測試 40
四、桑黃液態發酵菌絲體脂細胞抗氧化測定 40
第六章 討論 42
一、 抑制腫瘤細胞存活試驗 42
二、 細胞週期變化試驗 43
三、 細胞週期變化因子探討 44
四、 對乳癌細胞貼附之影響 44
五、 MMPs 活性試驗 45
六、 間接免疫與免疫能力活性試驗 45
七、 桑黃多醣之細胞內抗氧化測定 46
第七章 結論 47
第八章 未來方向 49
第九章 參考文獻 50



表目錄
表一、實驗中所使用到的抗體 18
表二、實驗中所使用到的引子(primer) 18
表三、以不同濃度之桑黃液態發酵菌絲體水萃物(F-W或M-W)處理MCF-7與MDA-MB-231細胞後細胞週期之變化表 35
表四、以5 mg/ml、10 mg/ml桑黃液態發酵菌絲體水萃物(F-W或M-W)處理MCF-7與MDA-MB-231 細胞24 hr後細胞週期之變化表 36
表五、以5 mg/ml、10 mg/ml桑黃液態發酵菌絲體水萃物(F-W或M-W)處理MCF-7與MDA-MB-231 細胞48 hr後細胞週期之變化表 36
表六、以5 mg/ml、10 mg/ml桑黃液態發酵菌絲體水萃物(F-W或M-W)處理MCF-7與MDA-MB-231 細胞72hr後細胞週期之變化表 37
表七、以5 mg/ml、10 mg/ml桑黃液態發酵菌絲體水萃物(F-W或M-W)處理MCF-7或MDA-MB-231 影響細胞週期因子之mRNA變化表 38
表八、以5 mg/ml、10 mg/ml桑黃液態發酵菌絲體水萃物(F-W或M-W)處理MCF-7或MDA-MB-231 影響細胞週期蛋白因子之變化表 39




圖目錄
圖一、 以不同碳源培養之液態發酵桑黃之發酵液處理乳癌細胞株MCF-7 72小時後對細胞株存活率之影響。 61
圖二、 以不同碳源培養之液態發酵桑黃之發酵液處理乳癌細胞株MDA-MB-231 72小時後對細胞株存活率之影響。 61
圖三、以不同碳源培養之液態發酵桑黃菌絲體水萃物處理乳癌細胞株MCF-7 72小時後對細胞株存活率之影響。 62
圖四、以不同碳源培養之液態發酵桑黃菌絲體水萃物處理乳癌細胞株MDA-MB-231 72小時後對細胞株存活率之影響。 62
圖五、以不同濃度液態發酵桑黃菌絲體水萃物(F-W)處理乳癌細胞株MCF-7 24hr後細胞週期之變化圖。 63
圖六、以不同濃度液態發酵桑黃菌絲體水萃物(M-W)處理乳癌細胞株MCF-7 24hr後細胞週期之變化圖。 64
圖七、以0至10.0 mg/ml液態發酵桑黃菌絲體水萃物(F-W)處理乳癌細胞株MDA-MB-231 24hr後細胞週期之變化圖。 65
圖八、以不同濃度液態發酵桑黃菌絲體水萃物(M-W)處理乳癌細胞株MDA-MB-231 24hr後細胞週期之變化圖。 66
圖九、以5 mg/ml或10 mg/ml液態發酵桑黃菌絲體水萃物(F-W或M-W)處理乳癌細胞株MCF-7 24 hr後細胞週期之變化圖。 67
圖十、以5 mg/ml或10 mg/ml液態發酵桑黃菌絲體水萃物(F-W、M-W)處理乳癌細胞株MCF-7 48 hr後細胞週期之變化圖。 68
圖十一、以5 mg/ml或10 mg/ml液態發酵桑黃菌絲體水萃物(F-W、M-W)處理乳癌細胞株MCF-7 72 hr後細胞週期之變化圖。 69
圖十二、以5 mg/ml或10 mg/ml液態發酵桑黃菌絲體水萃物(F-W、M-W)處理乳癌細胞株MDA-MB-231 24 hr後細胞週期之變化圖。 70
圖十三、以5 mg/ml或10 mg/ml液態發酵桑黃菌絲體水萃物(F-W、M-W)處理乳癌細胞株MDA-MB-231 48 hr後細胞週期之變化圖。 71
圖十四、以5 mg/ml或10 mg/ml液態發酵桑黃菌絲體水萃物(F-W、M-W)處理乳癌細胞株MDA-MB-231 72 hr後細胞週期之變化圖。 72
圖十五、桑黃液態發酵菌絲體水萃物(F-W或M-W)處理MCF-7細胞後細胞週期相關mRNA表現之電泳圖。 73
圖十六、桑黃液態發酵菌絲體水萃物(F-W或M-W)處理MDA-MB-231細胞後細胞週期相關mRNA表現之電泳圖。 74
圖十七、桑黃液態發酵菌絲體水萃物(F-W或M-W)處理MCF-7細胞後細胞週期相關蛋白質表現之電泳圖。 75
圖十八、桑黃液態發酵菌絲體水萃物(F-W或M-W)處理MDA-MB-231細胞後細胞週期相關蛋白質表現之電泳圖。 76
圖十九、不同濃度之液態發酵菌絲體水萃物(F-W或M-W)抑制MDA-MB-231細胞貼附試驗。 77
圖二十、桑黃液態發酵菌絲體水萃物在不同濃度對人類癌化纖維母細胞(HT1080)分泌MMP-2及MMP-9分解細胞外基質之活性影響 78
圖二十一、經不同濃度桑黃F菌絲體水萃物處理人類癌化纖維母細胞(HT1080) 24小時後MMP-2及MMP-9 mRNA表現 79
圖二十二、經不同濃度桑黃M菌絲體水萃物處理人類癌化纖維母細胞(HT1080) 24小時後MMP-2及MMP-9 mRNA表現 80
圖二十三、以不同碳源的桑黃發酵液處理Raw264.7細胞所得的上清液再處理癌細胞MCF-7後存活率之影響。 81
圖二十四、以不同碳源的桑黃發酵液處理Raw264.7細胞所得的上清液再處理癌細胞MDA-MB-231後存活率之影響。 81
圖二十五、以不同碳源的桑黃純化多醣處理Raw264.7細胞所得的上清液再處理癌細胞MCF-7後存活率之影響。 82
圖二十六、以不同碳源的桑黃純化多醣處理Raw264.7細胞所得的上清液再處理癌細胞MDA-MB-231後存活率之影響。 82
圖二十七、以RT-PCR偵測Raw264.7透過不同碳源桑黃發酵液誘導提升免疫因子的表現。 83
圖二十八、以RT-PCR偵測Raw264.7透過不同碳源桑黃純化多醣誘導提升免疫因子的表現。 83
圖二十九、桑黃菌絲體水萃物抗氧化能力(重疊圖);利用流式細胞儀偵測H2O2誘導60 min的CHO-K1細胞,使細胞產生大量ROS後給予桑黃菌絲體水萃物(M-W、L-W、S-W、F-W、D-W、G-W)測試清除自由基ROS的能力。 85
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