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研究生:陳佳鈺
研究生(外文):Chia-Yu Chen
論文名稱:固態栽培樟芝防治癌症之重要機轉-癌症逆轉
論文名稱(外文):Cancer reversion as a character of solid state cultivatedCancer reversion as a character of solid state cultivatedTaiwanofungus camphoratus for cancer prevention
指導教授:鄧文炳鄧文炳引用關係
指導教授(外文):Win-Ping Deng
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:生物醫學材料研究所
學門:工程學門
學類:生醫工程學類
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:80
中文關鍵詞:癌症逆轉子宮頸癌細胞腸鹼性磷酸酵素(IAP)致癌基因E6/E7抑癌基因p53樟芝
外文關鍵詞:Cancer ReversionIAPE6/E7p53HeLa cellTaiwanofungus camphoratus
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中文摘要

根據研究發現癌症是可以逆轉的,以感染病毒或以化學物質處理或與放射線接觸等方式藉著啟動腫瘤細胞中之逆轉作用機制,讓癌細胞不表現腫瘤特性,甚至於恢復成相當程度之正常細胞。所謂癌症逆轉現象(Cancer Reversion),意指腫瘤細胞回復「部分正常」,而成為不具威脅性地可與人體共存的人癌共生現象。
本實驗室已經建立了可供癌症逆轉機制探討的研究模式。樟芝﹐又稱牛樟芝或牛樟菇,是台灣特有的真菌,因無法以人工栽培,因此更顯得珍貴。以其中的多醣體(Polysccharides)、三萜類(Triterpenoids)及超氧歧化酵素(Superoxide Dismutase)為主要的研究份。根據初步研究樟芝有明顯的抑制腫瘤細胞生長及抑制癌病毒作用的效果,且以樟芝進行細胞試驗,結果顯示樟芝具有癌症逆轉的特性。
具腫瘤誘發性的HeLa細胞的表面抗原,其具有自磷酸化(Autophosphorylation)活性,是一種細胞的腸鹼性磷酸酵素(Intestinal alkaline phosphatase, IAP),因此本實驗室嘗試解析IAP表現與腫瘤誘發性之間的關係。實驗結果發現樟芝能夠抑制腫瘤細胞內鹼性磷酸酵素的表現。此外在基因層面的探討,初步實驗結果發現, 樟芝有增強抑癌基因p53表現的趨勢;而對子宮頸癌主要的E6/E7致病因子則有抑制的功效。在動物實驗方面,餵食樟芝對於腫瘤細胞生長確實有抑制的效果。以流式細胞儀技術分析樟芝對癌細胞的影響,可能並非走細胞毒殺的路徑,因此可以更加的推斷藥物對細胞的作用是進行癌症逆轉的模式。

關鍵字:癌症逆轉、子宮頸癌細胞、腸鹼性磷酸酵素(IAP)、致癌基因E6/E7、抑癌基因p53、樟芝
Abstract

Neoplastic transformation of normal cells can occur spontaneously or can be induced by viruses, chemical carcinogens, or irradiation. Such transformed cells; however, can also spontaneously, or be induced to, lose some of their tumorgenic characteristics and revert back to a more normal phenotypic state through correction of malignant behavior by microenvironmental cues. It has been suggested that the frequency of cell reversion to the non-tumorigenic phenotype is much higher than re-reversion to the tumorigenic phenotype, and that the reversion is paralleled by a regulation in malignant behavior and tumorigenicity. Such reversion may be a consequence of both genetic and epigenetic processes. One of the most challenging goals of cancer therapy is to exploit the potential of such reversion. Meeting this goal requires the identification of agent(s) with the ability to arrest tumor growth while having minimal effect on normal tissue. Taiwanofungus camphoratus, known as Niu-Chang-Chih or Niu-Chang-Ku, is rare and expensive. It growths only on the inner heartwood wall of the endemic evergreen Cinnamonum kanehirai and is not easily cultivated. The main ingredients of the fruit body includes: Polysaccharides; Triterpenoids and Superoxide dimutase(SOD). Traditionally, it is used as a Chinese remedy for food, alcohol, and drug intoxication, diarrhea, abdominal pain, hypertension, skin itches, and liver cancer. Concentrated ethanol extract of fruiting bodies of Taiwanofungus camphoratus exhibited immunomodulating effects in human leukocytes; exited antioxidant effects, and induced apoptosis in cultured human premyelocytic leukemia HL-60 cells. In addition Taiwanofungus camphoratus polysaccharides exhibit anti-hepatitis B virus effects. The expression of the HeLa tumor-associated marker, intestinal alkaline phosphatase (IAP), has been shown to be necessary but not sufficient to confer the tumorigenic phenotype on Hela x skin fibroblast human hybrid cells. We have previously used the loss of IAP activity and expression to investigate the reversion of a UVC-induced tumorigenic HeLa x skin fibroblast human hybrid cell line to the non-tumorigenic phenotype. Now, we describe for the first time a series of observations on the reversion of tumorigenic HeLa cervical carcinoma cells by Taiwanofungus camphoratus to a much less aggressive tumorigenic phenotype. This reversion was associated with the down-regulation of E6/E7 oncoproteins at the transcriptional level and subsequent reactivation of p53.

Key words: Cancer Reversion, IAP, E6/E7, p53, HeLa cell,
Taiwanofungus camphoratus
目錄

中文摘要……………………………………………………………………..I
英文摘要..........................................................................................................Ⅱ
第壹章 緒論......................................................................................................1
一、腫瘤的形成機轉....................................................................................2
二、子宮頸癌的介紹....................................................................................3
三、癌症逆轉的機制....................................................................................8
四、子宮頸癌癌症逆轉的研究....................................................................9
五、菇類與癌症的相關性..........................................................................10
六、樟芝的介紹..........................................................................................10
七、樟芝於子宮頸癌癌症逆轉機制研究應用於治療肺癌的動物模式..15
八、以樟芝應用於子宮頸癌癌症逆轉實驗設計流程..............................16
第貳章 材料與方法........................................................................................17
一、藥物與試劑..........................................................................................17
二、細胞株與動物......................................................................................19
三、藥物製備..............................................................................................19
四、細胞存活率試驗分析.............................................................................19
五、鹼性磷酸酵素分析..............................................................................20
六、腫瘤細胞回復率分析..........................................................................21
七、流式細胞儀技術..................................................................................21
八、蛋白質西方墨點法..............................................................................23
九、DNA聚合酶連鎖反應.........................................................................27
十、動物實驗..............................................................................................29
十一、原始細胞培養..................................................................................31
十二、蘇木紫與伊紅染色..........................................................................32
第參章 實驗結果............................................................................................33
一、細胞存活率試驗..................................................................................33
二、鹼性磷酸酵素分析..............................................................................33
三、腫瘤細胞回復分析..............................................................................33
四、流式細胞儀技術分析..........................................................................34
五、蛋白質西方墨點法分析......................................................................34
六、DNA聚合酶連鎖反應分析................................................................35
七、動物實驗..............................................................................................35
八、原始細胞培養......................................................................................36
九、蘇木紫與伊紅染色..............................................................................37
第肆章 討論....................................................................................................38
圖與表
圖一、樟芝成分之HPLC分析…………………………………………..…….43
圖二、細胞存活率試驗..............................................................................44
圖三、鹼性磷酸酵素分析..........................................................................45
圖四、腫瘤細胞回復分析..........................................................................46
圖五、流式細胞儀技術分析......................................................................47
圖六、蛋白質西方墨點法..........................................................................48
圖七、DNA聚合酶連鎖反應.....................................................................49
圖八、老鼠體內腫瘤的生長情況..............................................................50
圖九、原始細胞培養..................................................................................50
圖十、H&E stain及腫瘤組織之數位照片................................................53
參考文獻..........................................................................................................55
附錄
附錄一、A、E6病毒蛋白與P53結合會造成P53蛋白失去功能
附錄一、B、E7蛋白會和細胞的 E2F 酵素結合, 而導致癌症抑制基因Rb-E2F的控制細胞增值功能喪失.............................................64
附錄二、行政院衛生署統計民國九十二年國內女性癌症排名
子宮頸位置及結構圖示..............................................................65
附錄三、子宮頸細胞的癌症抑制基因失能,因而導致癌症抑制基因的功能喪失,進而產生變..................................................................66
附錄四、人類乳突瘤病毒基因體構造......................................................67
附錄五、牛樟芝外觀型態與特徵............................................................69
附錄六、MTT化學結構式.........................................................................70
細胞存活率試驗分析之實驗示意圖
附錄七、抗病毒藥物GCV的化學結構式.................................................71
抗病毒藥物GCV的作用機制
附錄八、樟芝應用於治療子宮頸癌癌的癌症逆轉模式實驗設計示圖...72
附錄九、樟芝應用於治療肺癌的癌症逆轉模式實驗設計示意圖……....73
參與發表的文獻:
Correction of malignant behavior of tumor cells by traditional Chinese herb medicine through a restoration of p53.
Cancer Letters (2005) 1–13
獲得九十三學年度師生聯合學術研究發表會優秀論文獎
壁報論文題目:
Induction Of Hela Cell Reversion By Taiwanofungus camphoratus, An
Indigenous Mushroom In Taiwan
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