臺灣博碩士論文加值系統

樟芝(Antrodia cinnamomea)是一種生長在牛樟樹上的蕈類，為台灣特有中藥，尤其具有抗腫瘤之功效。在本實驗室已證實樟芝發酵液(AC-10)具抗乳癌作用，會誘導乳癌細胞之細胞凋亡，但詳細機轉未探討。因此本論文將由細胞實驗(in vitro)和動物實驗(in vivo)兩方面來探討AC-10調控乳癌細胞株(MDA-MB-231)細胞週期與轉移之機制。
在細胞實驗中發現AC-10在劑量與時間效應上皆會降低乳癌細胞之Cyclin D1、CDK4、Cyclin E、Cyclin A和PCNA蛋白表現，增加P27和P21蛋白表現，同時AC-10可經由活化JNK pathway調控Cyclin D1表現，使細胞週期停滯在G0/G1期。本研究亦探討AC-10對MMP-9、uPA、tPA、PAI-1、TIMP-2及VEGF等乳癌細胞轉移相關因子之影響。結果由西方墨點法得知AC-10可減少MMP-9、uPA和VEGF蛋白表現，但會增加PAI-1及TIMP-2蛋白表現，以zymography assay也發現MMP-9、uPA和tPA之酵素活性受到抑制。另外進一步利用酵素連結免疫吸附法發現AC-10可抑制VEGF及tPA的分泌量，但會增加PAI-1的分泌量。
在動物實驗方面，給予注射AC-10之實驗組其腫瘤顯著小於控制組，利用免疫組織染色發現腫瘤組織中調控細胞週期演進之蛋白Cyclin D1、PCNA其抗原性顯著低於控制組，而與轉移相關之PAI-1蛋白其抗原性顯著高於控制組。另外，免疫組織染色也觀察到實驗組腫瘤組織Bcl-2和COX-2之表現低於控制組，進一步利用TUNEL assay發現給予AC-10之實驗組有細胞凋亡情形。
綜合上述細胞實驗和動物實驗之結果，證實AC-10藉由調控乳癌細胞之細胞週期蛋白表現而誘導細胞週期停滯，進而導致細胞凋亡。在腫瘤轉移方面AC-10會影響乳癌腫瘤轉移之相關酵素表現與活性，來抑制癌細胞之轉移。因此AC-10在未來醫藥之應用上可望成為具潛力的抗乳癌藥物。

Antrodia cinnamomea is well known in Taiwan as a traditional Chinese medicine. Plants possess many phytochemicals with various bioactivities , which include anticancer activities. In this laboratory analyzed the Antrodia cinnamomea fermentation fluid (AC-10) which had anti-breast cancer effect and could induce the breast cancer cell apoptosis, but mechanisms was unclear. In this study, we have addressed the cytostatic and apoptosis effects of AC-10 on breast cancer cell line (MDA-MB-231) in vitro and in vivo.
Treatment of MDA-MB-231 with AC-10 resulted in the inhibition of cell proliferation,arrested of cell cycle in G0/G1 phase and commitment to apoptosis. The results also demonstrated that down-regulate the expression of cell cycle progress factors Cyclin D1, CDK4, Cyclin E, Cyclin A and PCNA , and up-regulate the expression of cell cycle progress factors P27 and P21 in dose- and time-dependent. simultaneously AC-10 may activate JNK pathway to regulate Cyclin D1 expression, induce arrest of cell cycle in G0/G1 phase. This research also discusses treatment of MDA-MB-231 with AC-10 effect metastasis relevant factors of breast cancer cell as MMP-9, uPA, tPA, PAI-1, TIMP-2 and VEGF. The result also demonstrated that down-regulate the expression of MMP-9, uPA and the VEGF,and up-regulate the expression of PAI-1 and the TIMP-2. By zymography assay, the activity of MMP-9, uPA and tPA is suppressed. Furthermore by the Enzyme-Linked Immunosorbent Assay also analyzed AC-10 which suppressed VEGF and the tPA secretion, but can increase PAI-1 the secretion.
Our results revealed that treatment of AC-10 inhibited MDA-MB-231 tumor growth in experiments. Whereas, AC-10 did not show any side effects in these in vivo studies. By the Immunohistochemistry, in tumour regulate cell cycle protein of Cyclin D1, PCNA expression obviously is lower than the control group, but higher than the control group of PAI-1which its antigen with metastasis relevant. Moreover, the Immunohistochemistry also observes the experimental group tumor to Bcl-2 and the COX-2 is lower than the control group. By TUNEL assay , that induce apoptosis in experiment groups which injected AC-10.
Our results suggest that AC-10 induce arrest of cell cycle in G0/G1 phase.AC-10 also effect enzyme expression and metastasis in the breast cancer tumor, which suppress the metastasis of the cancer. Therefore, AC-10 had could be used as a health food preventing and alleviating MDA-MB-231 proliferation and possess the potential to be developed as an anticancer drug

目 錄
縮寫表-----------------------------------------------------------------Ⅰ
中文摘要--------------------------------------------------------------Ⅷ
英文摘要--------------------------------------------------------------Ⅹ
第一章、緒論---------------------------------------------------------1
第一節、細胞週期------------------------------------------------3
第二節、細胞凋亡------------------------------------------------9
第三節、活化絲裂原活化蛋白激酶家族(MAPKs)途徑與細胞凋亡-----------------------------------------------------15
第四節、環氧化酵素-2(COX-2)與乳癌之相關性----------18
第五節、腫瘤之侵入與轉移------------------------------------20
第六節、腫瘤動物模式簡介------------------------------------22
第七節、樟芝相關之文獻背景---------------------------------23
第八節、研究動機------------------------------------------------32
第二章、研究方法---------------------------------------------------34
第一節、研究器材------------------------------------------------34
第二節、細胞實驗------------------------------------------------37
第三節、動物實驗------------------------------------------------49
第三章、研究結果與圖表------------------------------------------55
第一節、樟芝(AC-10)對人類乳癌細胞株(MDA-MB-231)細胞週期之影響---------------------------------------------55
第二節、樟芝(AC-10) 活化MAPK pathway調控人類乳癌細胞株(MDA-MB-231)之細胞週期-------------------56
第三節、樟芝(AC-10)對於人類乳癌細胞株(MDA-MB-231)轉移相關酵素蛋白表現與活性之影響--------------57
第四節、樟芝(AC-10)對於移植人類乳癌細胞株(MDA-MB-231)的活體動物之影響---------------59
第四章、討論----------------------------------------------------------88
第五章、結論----------------------------------------------------------93
第六章、參考文獻----------------------------------------------------94


















圖表目錄
圖一、細胞週期調控圖示-------------------------------------------------8
圖二、細胞凋亡與細胞壞死的區別--------------------------------------10
圖三、細胞凋亡之訊息調控路徑---------------------------------------- 12
圖四、MAPKs 之訊息傳遞路徑圖---------------------------------------18
圖五、COX-2下游產物PGE2影響細胞生理機制----------------------------19
圖六、MMPs調控機制------------------------------------------------------------22
圖七、樟芝子實體實型態--------------------------------------------------------24
圖八.(A) MDA-MB-231以不同濃度之AC-10處理24小時，經流式細胞儀分析之細胞週期分佈---------------------------------------------------62
(B) MDA-MB-231經不同濃度之AC-10處理24小時，細胞週期分佈量化圖------------------------------------------------------------------63
圖九.以0、40、80、160、240 ?慊/mL AC-10處理24小時和以160 ?慊/mL AC-10處理後在0、4、8、12、18、24小時時間點分析MDA-MB-231細胞之Cyclin D1、Cyclin E、Cyclin A表現量------------------------64
圖十.以0、40、80、160、240 ?慊/mL AC-10處理24小時和以160 ?慊/mL AC-10處理後在0、4、8、12、18、24小時時間點分析MDA-MB-231細胞CDK4、CDK2、CDC2蛋白表現----------------------------------66
圖十一.以0、40、80、160、240 ?慊/mL AC-10處理2 4小時和以160 ?慊/mL AC-10處理後在0、4、8、12、18、24小時時間點分析MDA-MB-231細胞p21、p27、PCNA之相對表現量--------------------------------68
圖十二.MDA-MB-231經由160 ?慊/mL AC-10處理後在不同時間點分析MAPK family protein (ERK1/2、p38、JNK)及其磷酸化之表現-70
圖十三. MDA-MB-231以MAPK pathway培養1小時後，加入160 ?慊/mL AC-10培養4小時分析Cyclin D1蛋白表現---------------------------72
圖十四. MDA-MB-231以0、40、80、120、160、240 ?慊/mL AC-10處理24小時後對於MMP-9蛋白表現與活性之影響---------------------74
圖十五. MDA-MB-231以0、40、80、120、160、240 ?慊/mL AC-10處理24小時後對於uPA蛋白表現與活性之影響-------------------------75
圖十六. MDA-MB-231以0、40、80、120、160、240 ?慊/mL AC-10處理24小時後對於tPA活性與分泌量之影響-----------------------------76
圖十七. MDA-MB-231以0、40、80、120、160、240 ?慊/mL AC-10處理24小時後對於PAI-1蛋白表現和分泌量及TIMP-2蛋白表現量之影響-------------------------------------------------------------------------77
圖十八. MDA-MB-231以0、40、80、120、160、240 ?慊/mL AC-10處理24小時後對於VEGF蛋白表現和分泌量之影響--------------------79
圖十九.裸鼠移植人類乳癌細胞株(MDA-MB-231)以腹腔注射AC-10後體重和腫瘤體積變化之影響----------------------------------------------80
圖二十.裸鼠移植人類乳癌細胞株(MDA-MB-231)以腹腔注射AC-10後觀察腫瘤抑制效果----------------------------------------------------------81
圖二十一. AC-10對裸鼠乳癌腫瘤的cyclin D1變化--------------------------82
圖二十二. AC-10對裸鼠乳癌腫瘤PCNA之變化-----------------------------83
圖二十三. AC-10對裸鼠乳癌腫瘤PAI-1之變化------------------------------84
圖二十四. AC-10對裸鼠乳癌腫瘤Bcl-2之變化-------------------------------85
圖二十五. AC-10對裸鼠乳癌腫瘤COX-2之變化-----------------------------86
圖二十六. TUNEL assay觀察AC-10誘導乳癌腫瘤細胞凋亡現象 -------87
圖二十七.AC-10抑制乳癌生長機制圖---------------------------------------- 93
表一、Proximate composistion in Antrodia cinnamomea---------------------25表二、三萜類及多醣體有效成份說明------------------------------------------27

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