臺灣博碩士論文加值系統

根據世界衛生組織的預測，全球罹患癌症的人數在西元2030年將會超過1千2百萬人口以上，而在台灣根據行政院衛生署最新生命統計資料指出，癌症仍為100年國人十大死因之首；因此，尋找新抗癌藥物是當前重要且急迫的課題。牛樟芝是台灣特有的藥用真菌之一，目前已有不少研究著重在其解毒、抗菌、抗病毒、抗發炎、保護肝臟以及抗癌作用之探討，但因其生長緩慢且數量稀少，故被視為是極其珍貴之藥材。故本實驗則利用牛樟芝宿主-牛樟樹葉片萃取物針對人類食道癌細胞進行實驗，觀察牛樟葉萃取物抗癌之效應。
由實驗結果顯示牛樟葉萃取物對於人類食道癌細胞株BE-3 和 CE 81T/VGH其IC50約為20 μg/ml；我們在接下來的實驗中取低於IC50之治療劑量(5 μg/ml 和10 μg/ml)來進行細胞毒性、細胞型態、細胞增生以及藥物敏感性測試實驗，皆可發現牛樟葉萃取物對於人類食道癌細胞具有明顯之生長抑制作用。
而在探討抑制癌細胞轉移相關實驗中發現牛樟葉萃取物的確具有抑制人類食道癌細胞轉移及減低其入侵能力的現象，尤以BE-3細胞株較為明顯；再者我們利用Gelatin zymograpghy進一步探討可能的機制，實驗結果顯示牛樟葉萃取物能夠減低人類食道癌細胞中基質金屬蛋白酶的表現，故推論此萃取物可能是經由抑制基質金屬蛋白酶表現來抑制癌細胞轉移或入侵之能力。在另一個實驗中，我們檢測了加入牛樟葉萃取物治療後細胞的血管內皮細胞生長因子含量，發現此萃取物並無法抑制其表現，故推論可能的機制並非經由血管內皮生長因子及其接受器之訊號傳遞路徑。
最後，實驗探討牛樟葉萃取物能否誘導癌細胞發生細胞凋亡之現象，我們利用細胞染色的方式觀察到人類食道癌細胞在經由牛樟葉萃取物治療24小時候會出現典型的細胞質濃縮、以及細胞膜出現泡狀突起之細胞凋亡表現；而利用西方墨點法則發現牛樟葉萃取物對於人類食道腺癌細胞株(BE-3) 之p53蛋白表現量並無明顯提升之效果，但對於人類食道鱗狀上皮癌細胞株(CE 81T/VGH) 則可增加該蛋白之表現，故推論在不同細胞中牛樟葉萃取物誘導細胞凋亡之訊號傳遞路徑可能不盡相同。
綜合上述，我們觀察到牛樟葉萃取物在抑制人類食道癌細胞生長與抑制轉移和入侵能力具有相當優秀之表現，此萃取物的確具有成為好的癌症輔助治療藥物之潛力；在未來我們仍須更進一步地分離純化出可能的有效純物質並且針對其可能作用的分子機制做更為深入的探討。

According to the World Health Organization reports, by years 2030, more than 12 million people will be suffered from cancer. In addition, by the epidemiological data from department of health (DOH, Taiwan), cancer still is the most lethal cause of death. Thereby, the development of new therapeutics or agent to control cancer is urgently. Antrodia cinnamomea is a Taiwan-unique medicinal fungus that has been widely studied for its effects on detoxification, anti-inflammation, anti-bacterial, anti-virus, liver protection and anti-cancer activities. Because of the slow growing, it has been seen as a valuable medicinal herb. In this study, we investigated whether leaves extract from the host plant (Cinnamomum kanehirai Hayata ) of A. cinnamomea may have some anti-cancer effects.
The results indicate that the IC50 of the leaves extract for human esophageal cancer cell lines: BE-3 and CE 81T/VGH is 20 μg/ml, and we take 5μg/ml and 10μg/ml as the therapeutic doses for cytotoxicity, cell morphology, cell proliferation, and drugs sensitivity assay. It has shown rather significant inhibitory effects on the growth of human esophageal cancer cells.
And we found that the leaves extract also has inhibitory effects on human esophageal cancer cell migration and reduce its invasion capacity, especially in the BE-3 cells. According to the results of gelatin zymograpghy analysis , the leaves extract can reduce the expression of matrix metalloproteinases (MMPs) in human esophageal cancer cells.
Finally, we observed the leaves extract can induce the cell morphological changes and some apoptosis-associated cell death, including chromatin condensation and cell membrane blebbing. There is no significant enhance of p53 expression by western blot analysis for BE-3 cells, but the p53 protein level was over-expression in CE 81T/VGH cells. So that the apoptosis signaling pathway may be different in different cell lines.
Our results suggest that the leaves extract from Cinnamomum kanehirai Hayata has a very good performance in the ability to inhibit cell growth of human esophageal cancer and inhibition of cancer cells metastasis or invasion, and this extract does have a good potential for cancer adjuvant therapy. We still have further isolated and purified pure substances and for the possible molecular mechanism to do more investigations.

Table of contents
指導教授推薦書
口試委員審定書
國家圖書館論文著作授權書
長庚大學博碩士論文授權書
誌謝……………………………………………………………………v
中文摘要…………………………………………………………….vi
Abstract……………………………………………………………viii
Table of contents………………………………………………x
Abbreviations……………………………………………………xvi
Introduction
Malignant cancer.............................................1
Human esophageal cancer………………….......…………………………..3
Cancer metastasis……………………………………………………..........7
Matrix metalloproteinases (MMPs)………………………………….....…9
Apoptosis pathways…………………………………………………..........11
P53 functions and regulation of p53 expression…………………….12
Role of p53 in cancer………………………………………………….......13
Natural products and their bioactivity….....................14
Leaves extract from natural products………………………………....16
Cinnamomum kanehirai Hayata and Antrodia cinnamomea……..…...18
The development of natural anti-cancer agents……………………….20
Specific aims……………………………………………………….........……..23
Experimental design……………………………………………………..........25
Materials and methods Materials…………………………………………....26
A. cinnamomea strain and pre-culture for fermentation…………..28
Preparation of Antrodia cinnamomea mycelial fermentation broth…....29
Plant material………………………………………………………...29
Leaves extraction from Cinnamomum kanehirai Hayata…………….30
Cell culture……………………………………………………………31
Drugs treatment………………………………………………………32
Cell Morphology……………………………………………………...33
Cell viability assay……………………………………………………34
Cell proliferation assay……………………………………………….34
Drug sensitivity (Colony formation) assay…………………………...35
Wound healing test……………………………………………………36
Cell migration assay………………………………………………….36
Cell invasion assay…………………………………………………...37
Gelatin zymograpgy…………………………………………………..38
Preparation of conditioned medium………………………………….39
Measure of human VEGF level in cell culture supernatant…………..40
Liu’s staining………………………………………………………….41
Preparation of whole cell lysate……………………………………...41
Western blot analysis…………………………………………………42
Statistical analysis…………………………………………………….43
Results and discussion
Effect of C.K. -E2 treatment on inhibition of cell viability of BE-3 and
CE 81T/VGH cells…………………………………………………….44
Effect of C. K. -E2 and Taxol treatment on inhibition of viability of BE-3 and CE
81T/VGH cells………………………………………………..45
Effect of AC-MFB treatment on inhibition of cell viability of BE-3 and
CE 81T/VGH cells……….................................…….46
Effect of pure compounds extracted from Antrodia cinnamomea fruiting
bodies treatment on inhibition of cell viability…………………..…...47
Morphological changes in human esophageal cancer BE-3 and
CE 81T/VGH cells…………....................................................48.
C. K. -E2 inhibited proliferation of human esophageal cancer BE-3 and CE 81T/VGH
cells……………............................................................49
C. K. -E2 reduced colonies formation of BE-3 and CE 81T/VGH
cells…………………………………………………………………...49
The inhibition of wound healing in BE-3, CE 81T/VGH and EA. hy926 cells treated with C.
K. -E2…………………………………………....50
Effect of C. K. -E2 on migration ability of human esophageal cancer BE-3 and CE 81T/VGH
cells………………………………….….......51
Effect of C. K. -E2 on invasion ability of human esophageal cancer BE-3 and CE 81T/VGH
cells…………………………………….…...51
Active form of MMP-9 in human esophageal cancer cells suppressed by C. K .-
E2………………………………………………………………52
Human VEGF level is not down-regulated by C.K. -E2 treated in BE-3 and CE 81T/VGH
cells……………………………………………….52.
Modes of cell death induced by C. K. -E2……………………...……..53
Effect of C. K .-E2 on p53 expression in BE-3 and CE 81T/VGH cells………..….54
Discussion……………………………………………………………….55
Conclusion………………………………………………………………57
Future perspectives……………………………………………………...58
Table
Table 1………………………………………………………………...61
Figures and legends
Figure 1……………………………………………………………….62
Figure 2……………………………………………………………….64
Figure 3……………………………………………………………….67
Figure 4……………………………………………………………….69
Figure 5……………………………………………………………….71
Figure 6……………………………………………………………….73
Figure 7……………………………………………………………….76
Figure 8……………………………………………………………….78
Figure 9……………………………………………………………….80
Figure 10……………………………………………………………...82
Figure 11……………………………………………………………...84
Figure 12……………………………………………………………...86
Figure 13……………………………………………………………...88
Figure 14……………………………………………………………...91
Figure 15……………………………………………………………...93
Figure 16……………………………………………………………...95
Figure 17……………………………………………………………...97
Figure 18……………………………………………………………...99
Figure 19…………………………………………………………….101
Figure 20…………………………………………………….............103
Figure 21……………………………………………….....................106
Figure 22…………………………………………………………….109
Figure 23…………………………………………………………….111
Figure 24…………………………………………………………….113
Figure 25…………………………………………………………….115
Figure 26…………………………………………………………….117
Figure 27…………………………………………………………….119
References……………………………………………………...………121
Appendix……………………………………………………………….130

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