臺灣博碩士論文加值系統

牛樟芝為台灣特有昂貴真菌，僅生長於牛樟樹上。許多文獻指出其具有抗癌效果，本研究萃取牛樟葉，探討其是否具有抗癌效果，以取代昂貴的牛樟芝。
本研究萃取牛樟葉，探討其對人類內皮細胞株EA.hy926 (human endothelial cell line)及小鼠內皮細胞株SVEC 4-10 (mice endothelial cell line)是否具有抑制血管新生的效果，以及抑制遷移(migration)、癌細胞毒殺。此外，探討HA22T (Human hepatoma cell line)細胞凋亡(apoptosis)及細胞週期(cell cycle)是否停滯。
實驗結果發現，牛樟葉萃取液(CKH’ leaf extraction)對於EA.hy926、SVEC 4-10及HA22T細胞毒性試驗，皆能有效毒殺或抑制其生長。血管新生方面，牛樟葉不但能抑制EA.hy926的細胞遷移，也能抑制HA22T肝癌細胞株的轉移作用。利用流式細胞儀觀察細胞週期也發現有細胞凋亡的現象。此外，牛樟芝菌絲體發酵液作用於HA22T也發現有細胞凋亡現象，並和牛樟葉萃取液結果互相比較。

Antrodia camphorata are Taiwan-unique medicinal fungus that only live on Cinnamomum kanehirae Hayata. Many of literatures indicated that Antrodia camphorat had effective antitumor activity. The main purpose of these studies extract and expact Cinnamomum kanehirae Hayata’ leaf that it will be in place of AC-MFB (A. camphorata mycelia fermentation broth).
The results showed that Cinnamomum kanehirae Hayata’ leaf could suppress the growing of cell lines ( EA.hy926、SVEC 4-10 and HA22T ). When EA.hy926 and HA22T are treated by extraction of Cinnamomum kanehirae Hayata’ leaf, we find both of them apoptosis. In conclusions, Cinnamomum kanehirae Hayata’ leaf suppress not only endothelial viability, migration in EA.hy926, but also in HA22T.

目錄
指導教授推薦書 ………………………………………………………
口試委員審定書 ………………………………………………………
長庚大學授權書 ………………………………………………………iii
誌謝 ……………………………………………………………………iv
摘要…...……………………………………………………………….....v
英文摘要…………………………………………………..….………....vi
目錄………………………...……………………………...……………vii
圖目錄…………………………………………………………............xi表目錄…………………………………………………………............xiii第一章 緒論……………………………………………………………...1 1.1研究動機………………………...……………………………………1
1.2研究目的……………………………………………...………………2
1.3研究架構…………………………...…………………………………2
第二章 文獻回顧………………………………………………………3 2.1牛樟芝……………………………….………………………………..3
2.1.1牛樟芝生理活性成分………………………………………………3
2.1.2牛樟芝的療效……………….………………...……………………3
2.2牛樟…………………………………………………………………...6
2.3牛樟葉………………………………………………………………...6
2.3細胞週期……….…………………………………………….……….6
2.3.1細胞週期…………….……………………….……………………..6
2.3.2細胞週期調控…………………………….…………...……………7
2.4細胞凋亡……………………...………………………………………8
2.5惡性腫瘤…………………………….…………………...……….…..8
2.5.1腫瘤成長……………………………………………………………9
2.5.2腫瘤轉移…….…………………………………………………….10
2.6血管新生作用…………………………………………………….…11
2.6.1血管新生作用與癌症……………………………………………..12
2.6.2調整血管新生作用之相關因子………………………………......13
2.6.3血管內皮生長因子VEGF………………………….…….……….13
2.7缺氧與血管新生……….……………………………………………14
2.7.1缺氧誘導因子……………………………….…………………….15
2.7.2缺氧誘導因子調控機制…………………………………………..15
2.7.3缺氧誘導因子1α與癌症…………………………………………17
2.8抗血管新生療法與癌症治療……………………………………….17第三章 實驗材料與方法…………………………………………….…19
3.1牛樟葉濃縮液製備…..…………………………………………..….19
3.1.1牛樟葉萃取…………….………………………………………….19
3.1.2實驗藥品與儀器……………………………………………...…...19
3.1.3牛樟葉濃縮液之多醣體濃度測定(酚˙硫酸法)…………...…....19
3.2動物細胞培養………..………………………………………………20
3.2.1細胞株….…………………………………………………….……20
3.2.2培養基組成……….…………………………………………….…21
3.2.3實驗藥品與儀器………..………………………………………….21
3.2.4細胞培養方式…………….……………………………………….22
3.2.5繼代培養…………………….…………………………………….22
3.2.6動物細胞解凍與保存…………….……………………………….23
3.2.7動物細胞保存……………………….…………………………….23
3.3細胞毒性測試( MTT assay)……..……….…………………………23
3.3.1原理…………………………….………………………………….23
3.3.2實驗藥品與儀器………………………………………….……….23
3.3.3方法………………………………………………………………..24
3.4細胞週期 (cell cycle)……………………………….………………24
3.4.1實驗藥品與儀器…………………………………………………..24
3.4.2方法………………………………………………………………..25
3.5內皮細胞遷移能力測試 ( migration assay )……………………….26
3.5.1實驗藥品與儀器…………………………………………………..26
3.5.2方法…….………………………………………………………….26
3.5.3計算方式………………….……………………………………….27
第四章 結果與討論.................................................................................28
4.1牛樟葉濃縮液多醣體含量……….……………….……...……...….28
4.2牛樟葉濃縮液對內皮細胞之毒殺能力…………………………….28
4.3不同pH值的牛樟葉濃縮液對內皮細胞之毒殺能力……...………29
4.4不同萃取溫度的牛樟葉濃縮液對內皮細胞之毒殺能力…………..29
4.5細胞週期…………………………………………………………….29
4.6 牛樟葉對內皮細胞遷移之影響…………………………………...30
4.7 牛樟葉對肝癌細胞轉移之影響…………………………………...30
第五章 結論與未來目標……………………………………………….32
參考文獻………………………………………………………………...33


圖目錄
圖一 例年癌症死亡比率………………………………………..….…..41
圖二 牛樟葉.............................................................................................42
圖三 細胞週期調控…………………………………………………….43
圖四 缺氧誘導因子調控機制…………………..…………………...…44
圖五 酚-硫酸法之醣類檢量線………….……………...………...……45
圖六 牛樟葉萃取液對內皮細胞株EA. hy926細胞毒殺情形………...46
圖七 牛樟葉萃取液對內皮細胞株SVEC 4-10細胞毒殺情形………..47
圖八 牛樟葉萃取液對肝癌細胞株HA22T細胞毒殺情形…….……...48
圖九 牛樟芝菌絲體發酵液對肝癌細胞株HA22T細胞毒殺情形………………………………………………………………………...49
圖十 牛樟芝菌絲體發酵液對內皮細胞株SVEC 4-10細胞毒殺情形………………………………………………………………………...50
圖十一 牛樟葉萃取液在不同pH值下的細胞毒殺情形…..…………..51

圖十二 牛樟葉濃縮液在不同萃取溫度下的細胞毒殺情形…..…..….52
圖十三 牛樟葉萃取液作用於內皮細胞株EA. hy926 48小時後，細胞
週期情形………………………………………………………………...53
圖十四 牛樟葉萃取液作用於肝癌細胞株HA22T 48小時後，細胞
週期情形………………………………………………………………...54
圖十五 牛樟芝菌絲體發酵液作用於肝癌細胞株HA22T 48小時後，細胞週期情形……………………………………………………………...55
圖十六 牛樟葉萃取液Control組作用於內皮細胞株SVEC 4-10，細胞
遷移情形………………………………………………………………...56
圖十七 牛樟葉萃取液0.25X(0.625mg/ml)作用於內皮細胞株SVEC 4-10，細胞遷移情形……………………………………………….…...57
圖十八 牛樟葉萃取液0.5X(1.25mg/ml)作用於內皮細胞株SVEC 4-10，細胞遷移情形……………………………………………….…...58
圖十九 牛樟葉萃取液0.75X(1.875mg/ml)作用於內皮細胞株SVEC 4-10，細胞遷移情形……………………………………………….…...59
圖二十 牛樟葉萃取液1.5X(3.75mg/ml)作用於內皮細胞株SVEC 4-10，細胞遷移情形……………………………………………….…...60
圖二一 牛樟芝菌絲體發酵液Control組作用於肝癌細胞株HA22T，細胞遷移情形………………………………………………...……….…...61
圖二二 牛樟芝菌絲體發酵液2X(5mg/ml)作用於肝癌細胞株HA22T，細胞遷移情形………………………………………………...…….…...62


表目錄
表一 細胞凋亡與壞死比較………………………………………….....63
表二 牛樟葉萃取液作用於EA.h926 48小時後，細胞週期情形…..…64
表三 牛樟葉萃取液作用於HA22T 48小時後，細胞週期情形………65
表四 牛樟芝菌絲體發酵液作用於HA22T 48小時後，細胞週期情形.66
表五 牛樟葉萃取液作用於SVEC 4-10 4、8、12小時後，觀察細胞遷移抑制(%)情形………………………………………………………….67
表六 牛樟葉萃取液作用於HA22T 8、16、20小時後，觀察細胞遷移抑制(%)情形…………………………………………………………….68

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