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研究生:張恆嘉
研究生(外文):Heng-Jia Chang
論文名稱:ZC008對於抑制活化後之大鼠肝星狀細胞生長之有效成分萃取
論文名稱(外文):Growth Inhibitory Effect of ZC008 Active Extracts on ActivatedGrowth Inhibitory Effect of ZC008 Active Extracts on Activated Rat Hepatic Stellate Cells
指導教授:吳立真
指導教授(外文):Li-Chen Wu
學位類別:碩士
校院名稱:國立暨南國際大學
系所名稱:應用化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:87
中文關鍵詞:肝纖維化肝星狀細胞ZC008細胞存活率測試
外文關鍵詞:Liver fibrosisHepatic stellate cellsZC008MTT assay
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台灣的本土疾病─肝病。據估計台灣地區每年有五千多人死於肝癌,四千多人死於肝硬化,在國人的十大死因中慢性肝病及肝硬化排名第六。而且肝癌是男性惡性腫瘤死因的首位,是女性死因的第二位,僅次於肺炎。在肝臟受損的過程中,肝臟內的肝星狀細胞會被刺激而活化,進而轉化為肌成纖維細胞,而產生大量細胞外基質 (extracellular matrix, ECM),主要為膠原蛋白(collagen) ,產生與清除失去平衡時,則會導致肝纖維化的形成。而肝纖維化,是指各種慢性肝病向末期肝硬化發展過程中必經的病理過程,一個人只要患有慢性肝炎,帶有肝纖維化的症狀產生時,若不及時治療,使得症狀將持續發展,最後則會引起肝硬化而死亡。調查顯示,肝硬化佔據國際上疾病死亡病因的第六位。
利用中草藥與天然物來抑制肝纖維化的研究是從70年代中期開始,而在近年結合細胞生物學以及分子生物學技術上的應用,使得利用中草藥與天然物抗纖維化的研究能更加地深入。本篇論文將利用天然物ZC008的萃取物與肝星狀細胞共同進行培養,並偵測細胞存活率來得知ZC008萃取物對於抑制活化後之肝星狀細胞生長的有效程度。
因先前並無關於ZC008成分分析等文獻報導,故本論文將從ZC008當中分離出其有效抑制肝星狀細胞生長之化合物,接著藉由細胞存活率 (MTT assay) 偵測其生物活性,並以光譜分析以及化學方法來確定其結構。
我們經過實驗已證實,從ZC008的乙酸乙脂萃取層所分離出來的Compound A:Gallic acid 以及Compound B:Quercetin-3-O-galactoside對於抑制活化後的肝星狀細胞的生長有ㄧ定程度的效果。 I
接著將利用DAPI DNA labeling染色來觀察我們所分離出來的化合物誘導活化後的肝星狀細胞死亡之機轉為行細胞凋亡 (Apoptosis) 或是細胞壞死 (Necrosis) 。
同時我們也利用一些在結構上類似於Quercetin的黃酮類(Luteolin、Galangin、Rutin ) 對於抑制活化後肝星狀細胞生長效果進行比較,以期望能找出抑制肝星狀細胞生長的相對化學結構。
Liver disease is a common disease in Taiwan. Based on the statistic figures, there are more than 5,000 people died in liver cancer and 4,000 died in cirrhosis each year. Chronic liver diseases and cirrhosis are ranked the sixth of the ten leading causes of death in Taiwan. Moreover, liver cancer is the first leading cause of death of male malignant tumor, and is the second in female population, subsequent to the pneumonia. Hepatic stellate cells are activated while liver is damaged. These cells will further turn into myo-fibroblasts, which will produce lots of extra-cellular matrix (ECM), especially collagen, and may result in cirrhosis, the sixth death cause in the world.
The use of Chinese herbal medicine for liver fibrosis has been suggested since the middle age of the seventies. The discovery of herbal active components and the application of cell biology and molecular biology on the study of HSC growth inhibitory effect become significant recently. ZC008 is a traditional folk medicine for treating liver diseases. There is little literature on the analysis of chemical components of ZC008, and no literature discussing its active components on reducing liver fibrosis. In this study, the attempt to isolate HSC growth inhibitory active components from ZC008 was made.
The results demonstrated that gallic acid (compound A) and quercetin-3-O-galactoside (compound B) isolated from ethyl acetate extracts of ZC008 showed a significant inhibitory effect on the growth of serum activated HSCs based on MTT assay. Furthermore, results from DAPI DNA staining indicated that compounds A and B might induce apoptosis of HSC due to the fragmented nuclear. Besides, flavonoids with similar structure as quercetin such as luteolin, galangin, and rutin were compared on the HSC inhibition activities were carried out to locate the possible functional groups responsible for the inhibitory effect.
目 錄
頁數
中文摘要-------------------------------------------------------------------------------------I
英文摘要-----------------------------------------------------------------------------------III
目錄-----------------------------------------------------------------------------------------V
圖表目錄--------------------------------------------------------------------------------VIII
頁數
第一章 序論--------------------------------------------------------------------------------1
第一節 實驗背景及目的-------------------------------------------------------------1
第二節 傳統中草藥治療肝纖維化的研究----------------------------------------2
第三節 ZC008之簡介----------------------------------------------------------------2
第四節 肝纖維化的介紹-------------------------------------------------------------3
第五節 細胞死亡----------------------------------------------------------------------6
第六節 常用於肝纖維化用藥:Quercetinu以及Silymarin的簡介------------7
第七節 黃酮類成分對於抑制肝星狀細胞之效果-----------------------------11
第八節 研究動機--------------------------------------------------------------------11
第二章 實驗部份------------------------------------------------------------------------12
V
第一節 實驗材料--------------------------------------------------------------------12
一、 細胞來源-----------------------------------------------------------------12
二、 藥品試劑-----------------------------------------------------------------12
三、 實驗器材-----------------------------------------------------------------13
四、 實驗試劑-----------------------------------------------------------------15
第二節 實驗方法與步驟-----------------------------------------------------------18
一、 ZC008萃取與分離-----------------------------------------------------18
二、 成份結構鑑定之方法--------------------------------------------------19
三、 肝星狀細胞培養以及細胞存活率測試條件-----------------------20
四、 DAPI DNA labeling 染色---------------------------------------------23
五、 Luteolin, Galangin, Quercetin, Rutin, Compound A與Compound B之抑制效果比較以及有效濃度測試-----------------------------24
第三章 實驗結果與討論---------------------------------------------------------------25
第一節 細胞測試結果--------------------------------------------------------------25
一、 ZC008各萃取層對於抑制活化後肝星狀細胞生長活性之比較-----------------------------------------------------------------------------25
二、 從EA層分離出的fractions之MTT assay結果比較--------------25
三、 從E15~22分離出之fractions的MTT assay 結果----------------25
VI
四、 Compound A、Compound B與Quercetin之MTT assay結果比較--------------------------------------------------------------------------26
第二節 有效成分之結構鑑定-----------------------------------------------------27
一、 Compound A:3,4,5-Trihydroxy-benzoic acid (Gallic acid) 的結構鑑定--------------------------------------------------------------------27
二、 Compound B:Quercetin 3 -O-Galactoside的結構鑑定-----------29
第三節Compound A、Compound B 與Silymarin之有效濃度測試及活性
比較----------------------------------------------------------------------------32
第四節Luteolin、Galangin、Quercetin、Rutin、Compound A與
Compound B之有效濃度測試及活性比較-----------------------------32
第五節Luteolin、Galangin、Quercetin Rutin、Compound A與
Compound B細胞測試 之 DAPI 染色結果-------------------------33
第四章 結論------------------------------------------------------------------------------34
第五章 未來發展與期許---------------------------------------------------------------37
參考文獻-----------------------------------------------------------38
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