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研究生:黃瑀玟
研究生(外文):Yu-Wen Haung
論文名稱:樟芝超臨界水萃取物對乳癌細胞之生物活性探討
論文名稱(外文):The Bioactivity Study of theSupercritical Fluid Extraction fromAntrodia camphorata on BreastCarcinoma Cells
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:101
畢業學年度:100
語文別:中文
論文頁數:96
中文關鍵詞:樟芝超臨界萃取抗腫瘤抗轉移抗氧化
外文關鍵詞:Antrodia camphorataBaxBcl-2MMP-2MMP-9
相關次數:
  • 被引用被引用:2
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超臨界萃取技術具有低溫萃取、低黏性及高擴散能力之特性,能將對熱不穩
定之物質完整的萃取,且保留其活性成分,故能將樟芝所含的活性物質萃取。樟
芝又名牛樟芝或牛樟菇學名為Antrodia camphorata,是台灣特有真菌類,其生物
活性具有抗腫瘤, 抗發炎,抗氧化等作用。因此本研究主要以固態培養
樟芝菌絲體在不同壓力下之超臨界樟芝萃取進行生物活性探討。結果顯
示經由固態培養樟芝水萃取物及不同壓力下超臨界樟芝萃取粉末之水萃取物處
理後隨著濃度增加而對乳癌細胞(MCF-7、MDA-MB 231)增生之抑制率增加,能夠
顯著抑制乳癌細胞( MCF-7、MDA-MB 231)的生長,更進一步利用流式細胞儀於細
胞凋亡上發現,隨著濃度增加促使細胞走向凋亡的現象,此外,透過RT-PCR 發現,
在細胞凋亡路徑中,經由固態培養樟芝水萃取物及不同壓力下超臨界樟芝萃取粉
末之水萃取物處理後,Bcl-2 mRNA 的表現量隨著濃度增加明顯被抑制,而對Bax
mRNA 表現量明顯升高;於西方墨點法試驗中也發現會影響乳癌細胞內Bcl-2 、
Bax 蛋白質之表現量,導致細胞凋亡現象。在抑制轉移試驗中發現固態培養樟芝菌
絲體之水萃物以及經由不同壓力下超臨界樟芝萃取粉末之水萃取物,處理HT-1080
分泌基質金屬蛋白酶MMP-2 及MMP-9,明顯抑制效果,此外在RT-PCR 試驗中
也發現,經由樟芝水萃及經由不同壓力下超臨界樟芝萃取粉末之水萃取物處理
後,細胞MMP-2 及MMP-9 mRNA 表現也受到抑制。在DCFH-DA 抗氧化試驗中,
經由流式細胞儀發現固態培養樟芝菌絲體之水萃物及經由不同壓力下超臨界樟芝
萃取粉末之水萃取物,處理CHO-K1 細胞,能夠減少細胞內ROS 含量,具有抗氧
化活性。在提升免疫試驗中發現,利用固態培養樟芝菌絲體之水萃物及經由不同
壓力下超臨界樟芝萃取粉末之水萃取物,對Raw 264.7 巨噬細胞刺激後能促使細
胞分泌細胞激素而抑制乳癌細胞(MCF-7、MDA-MB 231 )增生。綜合以上結果證實
固態培養樟芝菌絲體之水萃物及經由不同壓力下超臨界樟芝萃取粉末之水萃取物確實有抗腫瘤、抑制轉移以及抗氧化之生物活性,實驗結果顯示樟芝及超臨界方
面值得開發。
The supercritical extraction technology, such as with low temperature extraction,
low viscosity and high diffusion capacity, has some benefit for food industry.
Therefore, these kinds of characteristics are good for unstable substances to retain their
active ingredient. Although many papers have shown that Antrodia camphorata has
several biological activities such as anti-tumor, anti-inflammatory, and anti-oxidation
effects, there are lots of biological functions still need to be elucidated. In order to
investigate the bioactivities of A. camphorata, the biological activities of the
supercritical fluid extraction (SFE) from A. camphorata under different pressures were
evaluated during this study. The results showed that the SFF from A. camphorata can
significantly inhibit the cell growth of breast cancer cells (MCF-7 and MDA-MB-231)
due to MTT assay. Moreover, the growth-inhibition effect in breast carcinoma cells
was due to apoptosis evaluated by flow cytometer. In addition, the pathway of
apoptosis on breast carcinoma cells was further confirmed by RT-PCR assay and
werstern blot anslyasis such as the Bax and Bcl-2 expression. Our data also showed
that SFE from A. camphorata has growth-inhibitory effect in tumor cells. In
antioxidant assay, we found that water extract or SFE of mycelia from A. camphorata
can reduce CHO-K1 cell death caused by H2O2. According immunomodulatory assay,
the results showed that the water extract or SFE can modulate cytokine production in
Raw 264.7 cells. Therefore, our data suggest that water extract or SFE from A.
camphorata have anti-tumor, anti-metastasis, antioxidant activity as well as
immunomodulatoy effect.
目 錄
中文摘要 i
英文摘要 iii
第一章 文獻回顧 1
一、研究背景 1
二、樟芝( Antrodia camphorata )簡介 1
2.1 特性 1
2.2 生理活性成分 2
三、超臨界流體 (Supercritical fluid,SCF) 3
3.1 超臨界流體發展 3
3.2 超臨界流體定義 3
3.3 超臨界流體萃取( Supercritical Fluid Extraction,SFE)基本原理及應用
4
四、癌症 5
4.1 乳癌的介紹 5
4.2 乳癌的治療 6
五、雌激素與雌激素受器 7
六、癌細胞的轉移與細胞凋亡 8
6.1 腫瘤轉移 8

6.2 細胞凋亡定義 9
6.3 細胞凋亡與細胞壞死 9
6.4 細胞凋亡之訊息調控路徑 10
第二章 研究動機 12
第三章 實驗架構 13
第四章 材料方法 14
一、實驗材料 14
1.1 實驗細胞株. 14
二、實驗方法 14
2.1 樣品製備 14
2.1.1 水萃取法 14
2.1.2 超臨界萃取方法 15
2.2 細胞存活率測定( MTT assay ) 15
2.2.1 實驗步驟 15
2.3 Gelatin Zymograpgy Assay 16
2.3.1Conditioned medium 與細胞溶解液(cell lysate)的收集 16
2.3.2 蛋白質含量的測定 17
2.4 電泳分析法 17
2.4.1 方法 17

2.5 細胞凋亡檢測 18
2.5.1 實驗步驟 18
2.6 西方墨點法(Western Bolt) 18
2.7 反轉錄聚合酵素鏈鎖反應 19
( RT-PCR,reverse transcription polymerase chain reaction)
2.7.1 mRNA 萃取 19
2.7.2 反轉錄聚合酶連鎖反應(RT-PCR) 20
2.7.3 電泳分析法cDNA 分析片段 ( Agarose gel electrophoresis ) 21
2.8 抗氧化能力測試 21
2.9 提升免疫能力測試. 21
2.9.1 實驗步驟 22
2.9.2 抑制癌細胞株生長試驗 22
2.10 統計分析 22
第五章 結果 24
一、抑癌試驗 24
1.1 抑制腫瘤細胞增生試驗 24
1.2 W、SFE-250 bar 及SFE-400 bar 對乳癌細胞之細胞凋亡影響 25
1.3 W、SFE-250 bar 及SFE-400 bar 對乳癌細胞Bcl-2、Bax mRNA 表
現之影響 27

1.4 W、SFE-250 bar 及SFE-400 bar 對乳癌細胞Bcl-2、Bax 、Pro-caspase-9
蛋白質表現之影響 27
二、轉移酵素試驗 28
三、W、SFE-250 bar 及SFE-400 bar 對細胞MMP-2 與MMP-9 m RNA 表
現的影響 28
四、W、SFE-250 bar 及SFE-400 bar 之細胞抗氧化測定 29
五、W、SFE-250 bar 及SFE-400 bar 免疫能力試驗 29
第六章 討論 32
一、抑制腫瘤細胞存活試驗 32
二、細胞凋亡試驗 32
三、樟芝水萃物(W、SFE-250 bar 及SFE-400 bar)對乳癌細胞Bax、Bcl-2 表
現之影響 33
四、MMPs 活性試驗 34
五、樟芝之細胞內抗氧化測定 34
六、間接免疫與免疫能力活性試驗 34
第七章 結論 35
第八章 未來方向 37
第九章 參考文獻 39

表目錄
表一、RT-PCR 分析所使用之primer 以及試驗的條件。 20
表二、W、SFE-250 bar、SFE-400 bar 對乳癌細胞(MCF-7、MDA-MB-231)存活
之影響。 24
表三、以不同濃度之樟芝及超臨界樟芝粉末水萃物(SFE-250 bar 及SFE-400 bar)
處理MCF-7 與MDA-MB-231 細胞12 hr 後細胞凋亡之變化。 26
表四、利用W、SFE-250 bar 及SFE-400 bar 刺激RAW264.7 細胞所得conditioned
medium 對MCF-7、MDA-MB-231 細胞存活之影響。 31

圖目錄
圖一、樟芝水萃取物(W)及超臨界二氧化碳樟芝粉末之水萃取物(SFE-250 bar、
SFE-400 bar) 對乳癌細胞(MCF-7)成長之影響。細胞處理不同濃度的W、
SFE-250 與SFE-400 bar (0 至8 mg/ml) 72 小時後之存活率。 52
圖二、樟芝水萃取物(W)及超臨界二氧化碳樟芝粉末之水萃取物(SFE-250 bar、
SFE-400bar) 對乳癌細胞(MDA-MB-231)成長之影響。細胞處理不同濃度的
W、SFE-250 與SFE-400 bar (0 至8 mg/ml) 72 小時後之存活率。 52
圖三、以樟芝水萃取物(W)及超臨界二氧化碳樟芝粉末之水萃取物(SFE-250 bar、
SFE-400 bar) MCF-7 細胞處理不同濃度的W、SFE-250 與SFE-400 bar (0
至8 mg/ml) 12 小時後細胞凋亡之變化圖。 55
圖四、以樟芝水萃取物(W)及超臨界二氧化碳樟芝粉末之水萃取物(SFE-250 bar、
SFE-400 bar) MDA-MB-231 細胞處理不同濃度的W、SFE-250 與SFE-400
bar (0 至8 mg/ml) 12 小時後細胞凋亡之變化圖。 58
圖五、以W處理MCF-7 細胞以不同濃度0、0.5、1、2、4、8 mg/ml, 12 小時後,
觀察Bcl-2、Bax mRNA 表現量。。。。。 59
圖六、以SFE-250 bar 處理MCF-7 細胞以不同濃度0、0.5、1、2、4、8 mg/ml,
12 小時後,觀察Bcl-2、Bax mRNA 表現量。 60
圖七、以SFE-400 bar 處理MCF-7 細胞以不同濃度0、0.5、1、2、4、8 mg/ml,
12 小時後,觀察Bcl-2、Bax mRNA 表現量。 61
圖八、以W處理MDA-MB-231 細胞以不同濃度0、0.5、1、2、4、8 mg/ml, 12
小時後,觀察Bcl-2、Bax mRNA 表現量。 62
圖九、以SFE-250 bar 處理MDA-MB-231 細胞以不同濃度0、0.5、1、2、4、8 mg/ml,
12 小時後,觀察Bcl-2、Bax mRNA 表現量。 63
圖十、以SFE-400 bar 處理MDA-MB-231 細胞以不同濃度0、0.5、1、2、4、8 mg/ml,
12 小時後,觀察Bcl-2、Bax mRNA 表現量。 64

圖十一、以W之不同濃度( 0、0.5、1、2、4、8 mg/ml ) 處理MCF-7 細胞24 小
時後,觀察Bcl-2、Bax 及pro-caspase-9 蛋白質之表現量。 65
圖十二、以SFE-250 bar 之不同濃度( 0、0.5、1、2、4、8 mg/ml ) 處理MCF-7
細胞24 小時後,觀察Bcl-2、Bax 及pro-caspase-9 蛋白質之表現量。 66
圖十三、以SFE-400 bar 之不同濃度( 0、0.5、1、2、4、8 mg/ml ) 處理MCF-7
細胞24 小時後,觀察Bcl-2、Bax 及pro-caspase-9 蛋白質之表現量。 67
圖十四、以W之不同濃度( 0、0.5、1、2、4、8 mg/ml ) 處理MDA-MB-231 細胞
24 小時後,觀察Bcl-2、Bax 及pro-caspase-9 蛋白質之表現量。 68
圖十五、以SFE-250 bar 之不同濃度( 0、0.5、1、2、4、8 mg/ml ) 處理MDA-MB-231
細胞24 小時後,觀察Bcl-2、Bax 及pro-caspase-9 蛋白質之表現量。69
圖十六、以SFE-400 bar 之不同濃度( 0、0.5、1、2、4、8 mg/ml ) 處理MDA-MB-231
細胞24 小時後,觀察Bcl-2、Bax 及pro-caspase-9 蛋白質之表現量。70
圖十七、以W處理人類癌化纖維母細胞(HT-1080)以不同濃度( 0、0.5、1、2、4、
8 mg/ml ) 24 小時後,觀察MMP-9、MMP-2 蛋白質之表現量。 71
圖十八、以SFE-250 bar 處理人類癌化纖維母細胞(HT-1080)以不同濃度( 0、0.5、
1、2、4、8 mg/ml ) 24 小時後,觀察MMP-9、MMP-2 蛋白質之表現量。
72
圖十九、以SFE-400 bar 處理人類癌化纖維母細胞(HT-1080)以不同濃度( 0、0.5、
1、2、4、8 mg/ml ) 24 小時後,觀察MMP-9、MMP-2 蛋白質之表現量。
73
圖二十、以W處理人類癌化纖維母細胞(HT-1080)以不同濃度( 0、0.5、1、2、4、
8 mg/ml ) 24 小時後,觀察MMP-9、MMP-2 mRNA 之表現量。 74
圖二十一、以SFE-250 bar 處理人類癌化纖維母細胞(HT-1080)以不同濃度( 0、0.5、
1、2、4、8 mg/ml ) 24 小時後,觀察MMP-9、MMP-2 mRNA 之表現
量。 75
圖二十二、以SFE-400 bar 處理人類癌化纖維母細胞(HT-1080)以不同濃度( 0、0.5、
1、2、4、8 mg/ml ) 24 小時後,觀察MMP-9、MMP-2 mRNA 之表現
量。 76
圖二十三、W、SFE-250 bar、SFE-400 bar 水萃物抗氧化能力(重疊圖);利用流式

細胞儀偵測H2O2 誘導60 分的CHO-K1 細胞,使細胞產生大量ROS 後
給予W、SFE-250 bar、SFE-400 bar 水萃物測試清除自由基ROS 的能
力。濃度為0.5、1、2 mg/ml 。 79
圖二十四、以W不同濃度(0.5、1、2、4、8 mg/ml)處理Raw 264.7 細胞所得上清
液再處理癌細胞MCF-7,72 小時後存活率之影響。 80
圖二十五、以SFE-250 bar 之不同濃度(0.5、1、2、4、8 mg/ml)處理Raw 264.7 細
胞所得上清液再處理癌細胞MCF-7,72 小時後存活率之影響。 80
圖二十六、以SFE-400 bar 之不同濃度(0.5、1、2、4、8 mg/ml)處理Raw 264.7 細
胞所得上清液再處理癌細胞MCF-7,72 小時後存活率之影響。 81
圖二十七、以W之不同濃度(0.5、1、2、4、8 mg/ml)處理Raw 264.7 細胞所得上
清液再處理癌細胞MDA-MB-231,72 小時後存活率之影響。 82
圖二十八、 以SFE-250 bar 之不同濃度(0.5、1、2、4、8 mg/ml)處理Raw 264.7 細
胞所得上清液再處理癌細胞MDA-MB-231,72 小時後存活率之影響。
82
圖二十九、 以SFE-400 bar 之不同濃度(0.5、1、2、4、8 mg/ml)處理Raw 264.7 細
胞所得上清液再處理癌細胞MDA-MB-231,72 小時後存活率之影響。
83
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