應用蛋白質體學技術探討松杉靈芝萃取物抑制人類卵巢癌細胞株SKOV-3生長之分子機轉; A proteomics approach to the molecular mechanism of growth inhibition by Ganoderma tsugae extracts in human ovarian cancer SKOV-3 cells

中國醫藥大學機構典藏 China Medical University Repository, Taiwan > 醫學院 > 醫學研究所 > 博碩士論文 > Item 310903500/1099

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題名:	應用蛋白質體學技術探討松杉靈芝萃取物抑制人類卵巢癌細胞株SKOV-3生長之分子機轉;A proteomics approach to the molecular mechanism of growth inhibition by Ganoderma tsugae extracts in human ovarian cancer SKOV-3 cells
作者:	王以均;Yi-Chun Wang
貢獻者:	中國醫藥大學：醫學研究所碩士班
關鍵詞:	松杉靈芝;生物反應指紋;卵巢癌細胞株SKOV-3;蛋白質體學;中草藥;Ganoderma tsugae;bioresponse fingerprinting;SKOV-3;proteomics;Traditional Chinese Medicine
日期:	2006-07-20
上傳時間:	2009-08-13 14:50:51 (UTC+8)
摘要:	靈芝在中國及東亞各國醫療保健和文化生活中的應用已有源遠流長的歷史。近年來，靈芝因其抗癌及免疫調節方面的功效而逐漸受到全球醫學界的重視。許多研究指出靈芝多醣具有促進免疫功能之活性，而靈芝三萜類化合物則能抑制腫瘤細胞生長，成為有潛力的抗癌藥物。本實驗室所使用的松杉靈芝子實體乾燥粉末經由薄層層析及高效液相層析技術鑑定其種源，且更進一步利用中草藥全球化聯盟主席鄭永齊院士所推廣的生物反應指紋分析平台，來監控靈芝萃取物之品質。結果顯示投予松杉靈芝甲醇萃取物的三組間之生物反應指紋圖譜極相似(PSI值>0.95)，而投予松杉靈芝甲、乙醇萃取物的兩大組間之生物反應指紋圖譜相似但不同。因此，生物反應指紋可作為中草藥品質管制的分析平台。本實驗室之前的實驗結果顯示，松杉靈芝之甲醇萃取物能抑制人類卵巢癌細胞株SKOV-3的生長。並且，根據創傷癒合實驗的結果，推測松杉靈芝甲醇萃取物亦能減緩SKOV-3細胞的移動能力。為闡明靈芝甲醇萃取物對於SKOV-3細胞造成諸多影響所牽涉的分子機轉，本論文利用蛋白質二維凝膠電泳分離，及介質輔助雷射脫附游離飛行時間式質譜儀等蛋白質體學技術，探討投予靈芝甲醇萃取物後SKOV-3細胞整體蛋白質表現的改變。結果得到51個Mascot評分值大於65，即p值小於0.05的蛋白質，其中，在投予靈芝甲醇萃取物後表現量明顯增加的蛋白質有22個，而明顯減少的蛋白質有23個。之後將其中40個蛋白質匯入GeneGo Meta CoreTM，進行訊號傳遞網絡圖的建立，並以西方墨點法輔證。最後，根據實驗結果以及相關文獻的探討，提出三個松杉靈芝甲醇萃取物抑制SKOV-3生長的可能訊號傳遞機轉：(1) 抑制c-Src、c-Myc及其下游基因的表現，而抑制了SKOV-3細胞的增生。(2) 影響HSP90及HSP27的正常功能，因而抑制SKOV-3細胞的增生及存活。(3) 抑制SKOV-3細胞醣解作用，因而降低細胞生長。本研究結果將可提供以中草藥對抗卵巢癌的可能細胞機轉與治療方法之參考依據。 Ganoderma, also known as Reishi or Lingzhi, has been used for medicinal purposes for centuries. This herb has gained considerable attention for its anti-tumor and immunomodulatory activities. We have investigated the extracts from the Ganederma tsugae (Gt), one of the Lingzhi species locally cultivated in Taiwan, followed by TLC and HPLC profiles analysis and further monitored by genetic bioresponse fingerprinting (PhytoViewerBR, PhytoCeutica, Inc.). The bioresponse fingerprinting results reveal that the PSI (Phytomics Similarity Index) value for the 3 independent experiments is very similar (PSI > 0.95), showing that the Gt extracts are in good quality. Moreover, our previous studies have shown that Gt methanol extracts have growth inhibition effects on the human ovarian cancer SKOV-3 cells, and decrease the oncogene expression. The wound closure assay result demonstrates that Gt methanol extracts also reduce SKOV-3 cells motility. To further unravel the molecular mechanism involved, a proteomics approach, 2-D electrophoresis and MALDI-TOF mass spectrometry, was used to tell the differential changes of protein expression profile in SKOV-3 cells after treatment with Gt extracts. Of the 51 noticeable altered protein spots with MOWSE Score>65 (i.e., p<0.05), 22 are up-regulated and 23 down-regulated. Forty characterized proteins were loaded on the GeneGo Meta CoreTM for building signaling network. Based on this signaling network followed by further Western blotting experiments, 3 possible molecular pathways leading to the growth inhibition of SKOV-3 cells by the Gt treatment have been proposed. Our results may provide possible cellular signaling networks of using the traditional Chinese medicine Ganoderma tsugae to treat ovarian cancer.
顯示於類別:	[醫學研究所] 博碩士論文

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