臺灣博碩士論文加值系統

摘要
牛樟芝(Antrodia camphorata)，僅生長在台灣特有的牛樟樹上，因數量稀少且久為民間所流傳之良好民俗偏方，成為台灣市場上昂貴的野生真菌。本論文之研究目的為樟芝菌絲體在體外模式中對HT-1080細胞株所分泌之金屬蛋白之影響進行探討。
以牛樟芝的水萃液當樣品進行研究，將HT-1080、MDA-MB-231、DU 145以及MCF-7等細胞，藉由MTT試驗、Gelatin zymography analysis及西方墨點法進行分析，檢視其對MMP-2與MMP-9蛋白質的影響，並用MMP-2與MMP-9的抗體做進一步的確認，另外，本實驗也以RT-PCR的方式進行樟芝菌絲體對MMP-2與MMP-9 mRNA 的表現之影響，並利用Transwell進行In Vitro Invasion Assay。
由實驗顯示樟芝萃取液對腫瘤細胞(HT-1080、MDA-MB-231、DU 145以及MCF-7)的抑制率分別為80%、74%、69%以及70%，此外在zymography analysis、抗體試驗及西方墨點法得到的結果可知，對HT-1080細胞之MMP-2與MMP-9分泌量有抑制效果，且在RT-PCR試驗中，初步結果有抑制MMP-2與MMP-9 mRNA 的表現，利用Transwell進行In Vitro Invasion Assay證明樟芝樣品可使癌細胞侵襲能力減少。
由結果證實樟芝萃取液對抑制腫瘤細胞是有效的，相同的對於HT-1080細胞之MMP-2與MMP-9分泌是有抑制的，且對腫瘤細胞的轉移也有抑制，因此很值得我們進一步研究。

ABSTRACT
Antrodia camphorata is a well-known fungus that only grows on Cinnamomum kanchirae and is only found in Taiwan with limited quantity.The aim of this study was to investigate the effect of the aqueous extract of Antrodia camphorata mycelium on MMP-2 and MMP-9 released from HT-1080 cells in vitro.
The biological activities of water extract of “niu-chang chih” were evaluated during this study. Furthermore, human tumor cells, such as MDA-MB-231, MCF-7, DU145 and HT1080, were used in this study. The biological activity of anti-tumor was assayed by MTT- microculture tetrazolium assay. Gelatin zymography analysis was used to assay the secretion of matrix metalloproteinases (MMPs), and the mRNA expression level was assayed by RT-PCR assay.
The results have shown that the inhibition cell growth rates of tumor cells (HT-1080, MDA-MB-231, DU145 and MCF-7) were 80%, 74%, 69%, 70%, separately. Furthermore, the results in Gelatin zymography analysis and RT-PCR have suggested that the protein and mRNA expression level of MMP-9 and MMP-2 on HT-1080 can be significantly inhibited after treated with water extract of “niu-chang chih”.
These results have demonstrated that there are very effective bioactivities of “niu-chang chih”, such as anti-tumor. According to the results of MMP-9 and MMP-2, it suggests that the water extraction of “niu-chang chih” may contribute to the inhibitory effect on tumor metastasis. Therefore, it is worthy to do further more studies on “niu-chang chih”.
Keywords : Antrodia camphorata, MMP, mycelia, invasion

目錄
中文摘要……………………………………………………………… I
英文摘要……………………………………………………………… II
目錄……………………………………………………………………III
圖目錄…………………………………………………………………VI
前言…………………………………………………………………… 1
文獻回顧……………………………………………………………… 2
一、 藥用菇的生理活性物質…………………………………… 2
1.多醣體……………………………………………………. 2
2.三帖類化合物……………………………………………. 3
3.超氧歧化酵素……………………………………………. 3
二、 樟芝介紹…………………………………………………… 4
三、 癌細胞轉移的過程………………………………………… 5
1.致癌基因的啟動…………………………………………. 5
2.新血管的形成……………………………………………. 6
3.癌細胞的剝離與移動……………………………………. 7
4.侵襲………………………………………………………. 9
5.脫離宿主的免疫系統…………………………………….12
6.吸附……………………………………………………….12
7.溢出……………………………………………………….13
8. 形成新的腫瘤…………………………………………...13
四、 研究動機……………………………………………………13
五、論文研究架構………………………………………………14
材料與方法……………………………………………………………15
實驗方法……………………………………………………………….19
一、 細胞繼代培養……………………………………………….19
二、 細胞冷凍保存……………………………………………….19
三、 冷凍細胞活化……………………………………………….20
四. 細胞計數與存活測試…………………………………………20
五. 細胞存活率測定………………………………………………20
六. Conditioned medium的收集與定量……………………………21 七. 電泳酵素分析法……………………………………………….22
八. 西方墨點法…………………………………………………….23 九. MMP-2、MMP-9 ELISA Assay………………………………..24
十. mRNA萃取……………………………………………………..25
十一. 反轉錄-聚合酵素鏈鎖反應…………………………………25
十二. 電泳分析法cDNA分析片斷……………………………….26
十三.移動試驗………………………………………………………27
十四.In Vitro Invasion assay………………………………………..27 十五.免疫試驗………………………………………………………28
十六.數據分析………………………………………………………28
實驗結果………………………………………………………………….29 一. 樟芝樣品對HT-1080、MCF-7、MDA-MB-231與DU-145 細胞增殖的討………………………………………………………..29
二. 樟芝萃取液對matrix metalloproteinase-2與matrix metalloproteinase-9 表現影響之探討……………………………….32三.以西方墨點法(Western blot)觀察不同樟芝萃取物處理HT-1080 細胞後，matrix metalloproteinase-2與matrix metalloproteinase-9 蛋白的表現…………………………………………………………..33
四、MMP-2與MMP-9 ELISA assay………………………………..34
五、探討樟芝萃取液對MMP-2與MMP-9 mRNA表現的影響…..35
六、探討HT-1080細胞經由樟芝萃取液處理後，對於細胞移動之影響……………………………………………………………………35
七、探討HT-1080細胞經由樟芝萃取液處理後，對於細胞轉移
(invasion)能力的影響…………………………………………37
八、探討RAW 264.7細胞經由樟芝萃取液處理後所分泌之物 質，對於HT-1080、DU-145、MCF-7與MDA-MB-231等細胞 之細胞增殖的影響………………………………………………37
討論…………………………………………………………………..39
參考文獻……………………………………………………………..95
附錄………………………………………………………………….103
圖目錄
圖一 : 樟芝水萃液處理HT-1080細胞存活的影響……………………43
圖二 : 樟芝水萃液處理MCF-7細胞存活的影響……………………..47
圖三 : 樟芝水萃液處理MDA-MB-231細胞存活的影響…………….50
圖四 : 樟芝水萃液處理DU-145細胞存活的影響……………………56
圖五 : 樟芝酒萃液處理AGS、DU145、HeLa、HT-1080和
MCF-7細胞存活的影響………………………………………..61
圖六 : HT-1080細胞以樟芝萃取液(AC-1)處理後MMP-2與
MMP-9分泌的影響……………………………………………..62
圖七 : HT-1080細胞以樟芝萃取液(AC-5)處理後MMP-2與
MMP-9分泌的影響……………………………………………..64
圖八 : HT-1080細胞以樟芝萃取液(子實體)處理後MMP-2與
MMP-9分泌的影響……………………………………………..66
圖九 : 樟芝萃取液(AC-1)對HT-1080細胞所分泌之MMP-2的
影響………………………………………………………………68
圖十 : 樟芝萃取液(AC-5)對HT-1080細胞所分泌之MMP-2的
影響………………………………………………………………69
圖十一 : 以MMP-2抗體探討樟芝萃取液處理HT-1080細胞後
MMP-2的分泌量…………………………………………………70
圖十二 : 以MMP-9抗體探討樟芝萃取液處理HT-1080細胞後
MMP-9的分泌量…………………………………………………75
圖十三 : 以RT-PCR探討樟芝萃取液AC-1處理HT-1080細胞後
分泌MMP-2與MMP-9 mRNA之表現…………………………..80
圖十四 : 以RT-PCR探討樟芝萃取液AC-1+PMA處理HT-1080
細胞後分泌MMP-2與MMP-9 mRNA之表現………………….81
圖十五 : 以RT-PCR探討樟芝萃取液AC-5處理HT-1080細胞後
分泌MMP-2與MMP-9 mRNA之表現…………………………82
圖十六 : 以RT-PCR探討樟芝萃取液AC-5+PMA處理HT-1080
細胞後分泌MMP-2與MMP-9 mRNA之表現…………………83
圖十七 : 樟芝萃取液處理HT-1080細胞後對細胞移動的影響……..84
圖十八 : 樟芝萃取液處理HT-1080細胞後對細胞轉移能力的影響..86
圖十九 : RAW 264.7細胞經由樟芝萃取液處理後所分泌之
物質，對於HT-1080細胞之細胞增殖的影響………………...87
圖二十 : RAW 264.7細胞經由樟芝萃取液處理後所分泌之
物質，對於MCF-7細胞之細胞增殖的影響…………………..89
圖二十一 : RAW 264.7細胞經由樟芝萃取液處理後所分泌
之物質，對於MDA-MB-231細胞之細胞增殖的影響………91
圖二十二 : RAW 264.7細胞經由樟芝萃取液處理後所分泌
之物質，對於DU-145細胞之細胞增殖的影響………………93

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