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研究生:謝蕙霞 
研究生(外文):Hui-Hsia Hsieh
論文名稱:以微晶片實驗探討天生靈芝乙醇萃液對非小細胞肺癌細胞株的抗癌作用
論文名稱(外文):A Gene Chip Approach to Anti-cancer Effect of Tien-Shen Lingzhi Ethanol Extracts on Non-Small Cell Lung Cancer Cell Line
指導教授:吳家樂高銘欽高銘欽引用關係
指導教授(外文):Ka-Lok NgMing-Ching Kao
學位類別:碩士
校院名稱:亞洲大學
系所名稱:生物資訊學系碩士班
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:65
中文關鍵詞:天生靈芝松杉靈芝微晶片肺癌細胞週期
外文關鍵詞:Tien-Shen LingzhiGanoderma tsugaeMicroarrayLung CancerCell Cycle
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背景:肺癌是所有惡性腫瘤疾病中預後最差的疾病之一,仍必須尋求更多的治療方法以改善臨床預後。從傳統中草藥尋找具有生物活性之治療成分已經成為一種新趨勢。幾個世紀以來,靈芝一直是廣被使用作為促進健康的中草藥。我們曾經發表過關於靈芝在不同類型癌症的抗癌功效,本研究以基因體實驗進一步探討天生靈芝(台灣松杉靈芝改良品種)抗癌作用相關的訊息傳導路徑。方法:以Affymetrix Human Genome U133 Plus 2.0 晶片設計實驗,藉由生物資訊工具GeneGo Meta CoreTM 分析比較,尋找非小細胞肺癌細胞株H23/0.3 經天生靈芝處理與否所引起的差異性表達基因。結果:非小細胞肺癌細胞株H23/0.3接受天生靈芝處理與否的全基因體表達, 由多元尺度分析(multidimentional scaling)呈現明顯之差異。生物資訊分析顯示,天生靈芝擾亂了非小細胞肺癌細胞株的細胞週期,且細胞週期調控相關的訊息傳導路徑是最主要被影響之標的。在26 個與DNA 合成相關的訊息傳導分子中,有17 個在本次分析中呈現差異。針對部分差異表達基因以Q-PCR 實驗驗證,結果亦與晶片實驗結果一致。再以流式細胞儀檢測,顯然地,經天生靈芝處理之肺癌細胞株呈現細胞週期停滯。結論:微晶片實驗顯示,天生靈芝擾亂非小細胞肺癌細胞株H23/0.3 的細胞週期,流式細胞儀檢測到的細胞週期擾亂與晶片生物資訊分析結果是一致的。
Background: Lung cancers are among the neoplastic diseases with the worst prognosis. More treatment modalities are needed to improve clinical outcome. It’s being a new trend to search for bioactive traditional Chinese medicines (TCM). Ganoderma, also known as Lingzhi, is a TCM and has been widely used for improving human health for centuries. We have addressed the anticancer effects of Ganoderma on various cancers. In this study, a comprehensive genomic profiling was used to further explore the signal pathway for the anticancer effects of Tien Shen Lingzhi(TSL),an improved strain of Ganoderma tsugae. Methods: A comparative genomics study was performed to search for TSL-mediated differential expressions on H23/0.3 cells. Bioinformatics tool, e.g., GeneGo Meta CoreTM ,was used to analyze the experimental results based on Affymetrix Human Genome U133 Plus 2.0 microarray. Results: The whole-genome expression profiles of H23/0.3 with or without TSL treatment were resolved by multidimentional scaling (MDS) analysis. It indicated a TSL-mediated cell cycle perturbation. Bioinformatics analysis showed cell cycle was the most affected pathway. GeneGo Meta CoreTM derived cellular pathway map revealed dramatic hits (17/26) on the signal transduction of DNA replication. The microarray results were confirmed by Q-PCR. Conclusion: The genomic microarray data demonstrates the TSL-induced cell cycle deregulation in H23/0.3 cells. TSL-mediated cell cycle perturbation on H23/0.3 cells is consistent with the cellular pathway map resulting from GeneGo Meta CoreTM analysis.
誌謝 -------------------------------------------------I
摘要 -------------------------------------------------Ⅱ
Abstract ---------------------------------------------Ⅲ
目錄 -------------------------------------------------Ⅳ
表目錄 -----------------------------------------------IV
圖目錄 -----------------------------------------------Ⅶ
第一章 緒論 -----------------------------------------1
第一節 研究背景 ----------------------------------1
一、 靈芝的分類 ---------------------------------2
二、 靈芝外觀 -----------------------------------3
三、 靈芝解剖 -----------------------------------3
四、 中國靈芝分類及考証 -------------------------4
五、 臺灣常見靈芝種類及特性----------------------6
六、 靈芝的成分 ---------------------------------7
七、 靈芝的抗癌研究 -----------------------------9
第二節 研究動機 ----------------------------------11
第三節 研究目的 ----------------------------------11
第二章 材料與方法 -----------------------------------12
第一節 研究材料 ----------------------------------12
一、 天生靈芝 -----------------------------------12
二、 天生靈芝萃液 -------------------------------12
三、 肺癌細胞株 ---------------------------------12
四、 微晶片 -------------------------------------13
五、 研究策略 -----------------------------------13
V
第二節 研究方法 ---------------------------------13
一、 細胞培養 ----------------------------------13
二、天生靈芝萃液抑制H23/0.3的細胞生長 ----------14
三、微晶片檢測分析 -----------------------------16
四、細胞週期分析 -------------------------------17
第三章 研究結果 ------------------------------------19
一、 肺癌細胞生長曲線 --------------------------19
二、 天生靈芝萃液抑制肺癌細胞生長 --------------20
三、 天生靈芝萃液對肺癌細胞基因體的影響 --------22
四、 天生靈芝抗癌作用細胞路徑分析 --------------27
五、 天生靈芝萃液抑制肺癌細胞之細胞週期 --------34
六、 比較 Q-PCR 與微晶片實驗結果 ---------------37
第四章 討論 ----------------------------------------41
一、 結果討論 ----------------------------------41
二、 相關討論 ----------------------------------43
三、 研究限制 ----------------------------------45
第五章 結論 ----------------------------------------46
參考文獻 --------------------------------------------47
表目錄
表一 Fold-Change 組合的相關係數 -----------------------22
表二 Primer sequence for real-time PCR ----------------38
圖目錄
圖1 肺癌細胞 H23/0.3 生長曲線圖 ------------------------19
圖2a H23/0.3肺癌細胞型態圖 ------------------------------20
圖2b 靈芝萃液對H23/0.3肺癌細胞的生長抑制曲線 ------------21
圖3 Volcano plot ---------------------------------------23
圖4 MDS (multidimentional scalin) plot (for all) -------24
圖5 MDS and Heat Map (absolute fold change > 1) --------25
圖6 MDS and Heat Map (0.6<absolute fold change<1.2) ----26
圖7 細胞功能性路徑對應 ---------------------------------28
圖8 Map for role of APC in cell cycle regulation -------29
圖9 Map for chromosome condensation in prometaphase ----30
圖10 Map for start of DNA replication in early S phase --31
圖11 Map for transition and termination of DNA replication--32
圖12 Map for the metaphase checkpoint -------------------33
圖13 天生靈芝萃液影響肺癌細胞株H23/0.3的細胞週期分析-Ⅰ--35
圖14 天生靈芝萃液影響肺癌細胞株H23/0.3的細胞週期分析-Ⅱ--36
圖15 比較 Q-PCR 與微晶片實驗結果 ------------------------40
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