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研究生:彭瓊琦
研究生(外文):Chiung-Chi Peng
論文名稱:利用細胞株探討樟芝對人類膀胱癌之療效與其作用機轉
論文名稱(外文):Characterization of Therapeutic Potential and Action Mechanism of Antrodia camphorata on Human Bladder Cancers through Cancer Cell Lines
指導教授:蘇慶華蘇慶華引用關係
學位類別:博士
校院名稱:臺北醫學大學
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文出版年:2007
畢業學年度:95
語文別:英文
論文頁數:114
中文關鍵詞:樟芝另類輔助療法膀胱癌細胞株
外文關鍵詞:Antrodia camphorataComplementary and Alternative Medicine (CAM)bladder cancer cells (RT4TSGH-8301and T24)
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膀胱癌是一種和環境或職業因素有關的惡性腫瘤,其中以移行性惡性細胞腫瘤(Transitional Cell Carcinoma)最為常見。在男性泌尿生殖系統的癌症死亡原因中居第二大位,而在台灣目前位居第六位,而且也有逐年上升的趨勢。
樟芝 (Antrodia camphorata) 是一種獨特而且具有許多醫療用途的菇菌植物,只生長在台灣特有的牛樟樹上,因此屬於稀少且昂貴的保育類植物之一。長久以來,曾經被用來當做藥物中毒、腹瀉、腹痛、高血壓及肝癌等的治療處方。基於近年來包括中草藥治療的另類輔助療法(CAM, The Complementary and Alternative Medicine) 逐漸興起,因此,我們嘗試探討樟芝是否具有抑制病理上惡性程度不同的膀胱癌細胞生長之作用,並進一步希望了解其作用機制,以作為臨床上使用的參考。藉由三株分化程度不同的膀胱癌細胞株 (RT4, TSGH-8301, 及T24)作為實驗組以及另一株老鼠胚胎纖維母細胞 (MEF) 作為正常細胞對照組,在加入樟芝子實體(fruit body)粗萃取物處理後的24至 72小時,利用MTT 呈色試劑分析,檢測其細胞存活率;以流式細胞儀分析細胞週期的分佈,並以西方墨點法針對其細胞週期相關的蛋白質的表現作分析;最後,則以一些和癌症轉移有關的實驗做功能性的探討。由初步的實驗結果顯示,不論是淺層侵犯性的膀胱癌細胞(例如 RT4),或是深層侵犯性的膀胱癌細胞(例如 T24),樟芝皆有抑制其生長的作用,惟其作用機制則因細胞不同而有所差別。RT4細胞經100 ?慊/mL樟芝粗萃取物處理24小時後,和G1週期相關的p21蛋白質表現明顯增加,同時,磷酯化的Rb蛋白質表現則逐漸減弱,此種變化,由於並無p53蛋白質的參與,應屬於細胞複製性的靜止現象 (replicative senescence);另一方面,TSGH-8301和T24細胞株以50 μg/mL樟芝粗萃取物處理48小時後,其和G2週期相關的Cdc2 及cyclin B蛋白質表現均逐漸減弱,且其移轉能力也被抑制,但正常的MEF細胞則未受樟芝粗萃取物之影響。
總而言之,對於復發性高或惡性轉移能力高的膀胱癌細胞,樟芝皆可能有其臨床醫療上之價值。
Bladder cancer has been cited to result from the neoplastic lesion with environmental and/or occupational factors identified as causatives. Among which the transitional cell carcinoma (TCC) is the most common. The mortality of TCC has now become the second top among the cancers of male urinary system, and overall, it is the top sixth cancer mortality in Taiwan, and still continuing to increase.
Antrodia camphorata (Polyporaceae) (AC), a unique mushroom that grows merely on the inner bark of a characteristic species of camphor tree called Cinnamomum kanehirai, is a very rare and valuable ecologically protected plant. Since long ago, it has been adopted for treatment of intoxication, diarrhea, abdominal pain, hypertension, and hepatoma. Recently the development of the so-called the Complementary and Alternative Medicine (CAM) has strongly attracted us to investigate the potential of anti-bladder cancer of AC. Tests were performed to check whether AC is capable of inhibiting the growth of bladder cancers of different stages, in addition, to understand the action mechanism of AC to ensure an effective and safe clinical use in the future. In this present study, we used three bladder cancer cell lines (RT4, TSGH-8301, and T24) and the mouse embryonic fibroblast cells (MEF) as the control cells. After treatment with the crude extracts obtained from the fruit bodies of AC for 24 and 48 h respectively, the cell viability was examined by MTT assay. Flow cytometric and Western blot analyses were conducted to study the cell population during the cell cycle and the expression of cell-cycle-related proteins. Migration capability of cancer cells affected by AC treatment was also determined by wound scratch and transwell analyses. Results from this study indicate that AC is capable of inhibiting all type of bladder carcinoma cells, despite of lower (e.g. RT4) or higher (e.g. T24) invasiveness. However, the action mechanisms are different among these cells. After treated with crude extract of AC (ACCE) at a concentration of 100 μg/mL for 24 h, the expression of G1 phase associated p21 protein was significantly up-regulated in RT4 cells In the meanwhile, the phosphorylated Rb was steadily down regulated in this high invasive cells. Being not participated by the protein p53, such a phenomenon was ascribed due to a phenomenon called replicative senescence. In contrast, after treated with ACCE at a concentration of 50 μg/mL for 48 h, Cdc2 and cyclin B, the proteins associated with the G2 phase in cells TSGH-8301 and T24, were all gradually downregulated. At the same time, the migration capability of these low invasive cells was completely suppressed. As a control, the MEF cells were totally unaffected.
In conclusion, the mushroom Antrodia camphorata might be effective for inhibiting all bladder cancer cells despite of their invasiveness, implicating its potential clinical application.
TABLE of CONTENTS
Page
表目次 (List of Tables)
................................................................................................................ 5
圖目次 (List of Figures)
................................................................................................................ 6
縮寫表 (Abbreviations)
................................................................................................................ 7
中文摘要 (Abstract in Chinese)
................................................................................................................ 9
英文摘要 (Abstract in English)
............................................................................................................... 11
第一章 文獻回顧 (Literature Review)
............................................................................................................... 14
第一節 膀胱癌
Bladder Cancers (BC)
............................................................................................................... 15
第二節 另類輔助療法
Complementary and Alternative Medicine (CAM)
............................................................................................................... 28
第三節 樟芝
Antrodia camphorata (AC)
............................................................................................................... 32
第三節之一 習性與敘述
Habitant and Description
............................................................................................................... 32
第三節之二 分類法
Taxonomy
............................................................................................................... 33
第三節之三 生物活性
Biological activities
............................................................................................................... 35
第四節 結語
Closing remarks
............................................................................................................... 36
第二章 材料與方法 (Materials and Methods)
............................................................................................................... 38
第一節 樟芝粗萃物的來源
The Source of Antrodia camphorata Crude Extract (ACCE)
............................................................................................................... 39
第一節之一 樟芝粗萃物組成分析
Compositional Analysis of ACCE
............................................................................................................... 39
第二節 化學藥品 Chemicals
............................................................................................................... 40
第三節 抗體 Antibodies
............................................................................................................... 42
第四節 儀器 Apparatus
............................................................................................................... 42
第五節 方法 Methods
............................................................................................................... 43
第五節之一 細胞培養 Cell Cultures
............................................................................................................... 43
第五節之二 細胞增生實驗
Cell Proliferation Assay
............................................................................................................... 44
第五節之三 掃描式電子顯微鏡顯影
Scanning Electron Microscopy
............................................................................................................... 45
第五節之四 流式細胞儀分析
Flow Cytometric Analysis
............................................................................................................... 46
第五節之五 西方墨點分析
Western Blot Analysis
............................................................................................................... 46
第五節之六 刮痕癒合實驗
Wound Scratch Assay
............................................................................................................... 48
第五節之七 癌細胞移動能力實驗
Transwell Motility Assay
............................................................................................................... 58
第五節之八 蛋白水解酶活性試驗
Zymography
............................................................................................................... 49
第五節之九 統計分析法
Statistical Analysis
............................................................................................................... 50
第三章 結果與討論 (Results and Discussion)
............................................................................................................... 51
第四章 結論與展望 (Conclusions and Perspective)
............................................................................................................... 64
圖表 (Tables and Figures)
............................................................................................................... 67
參考文獻 (References)
............................................................................................................... 88
附錄 (Appendix) --- 論文選集
(Peer-Reviewed Articles)
...............................................................................................................114
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