跳到主要內容

臺灣博碩士論文加值系統

(44.211.117.197) 您好!臺灣時間:2024/05/27 05:38
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:林怡慧
研究生(外文):Yi-Huei Lin
論文名稱:Gamma-tocotrienol在肝癌細胞模式中抑制膽固醇合成路徑探討
論文名稱(外文):Mechanistic studies on the cholesterol synthesis inhibition by gamma-tocotrienol in HepG2 cell model
指導教授:黃卓治黃卓治引用關係
指導教授(外文):Tzou-Chi Huang
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:生物科技系所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:59
中文關鍵詞:HMG CoA reductase膽固醇Gamma-生育三烯醇monacolin K異戊二烯化Ras
外文關鍵詞:HMG-CoA reductasecholesterolγ-tocotrienolmonacolin KisoprenylationRas
相關次數:
  • 被引用被引用:0
  • 點閱點閱:231
  • 評分評分:
  • 下載下載:44
  • 收藏至我的研究室書目清單書目收藏:0
二羥甲戊酸途徑(mevalonate pathway)速率控制酵素為HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, HMGR),經由此代謝途徑可產生膽固醇。Gamma-生育三烯醇(γ-tocotrienol) 化學構造與金合歡醇類似,故推測γ-tocotrienol可促進 HMG-CoA reductase之降解,本研究是以人類肝癌細胞株(HepG2)為研究模式,探討測試γ-tocotrienol對膽固醇生合成關鍵酵素HMG-CoA reductase抑制機制探討。紅麴含有一些化合物統稱為monacolins,為抑制膽固醇合成的物質。其中之一為monacolin K,是HMG-CoA還reductase的抑製劑,也稱為mevinolin或洛伐他汀(lovastatin)。根據西方墨點法得知γ-tocotrienol抑制HMG-CoA reductase蛋白表現,γ-tocotrienol 及 monacolin K隨著劑量的增加可減少總膽固醇生成,再以共軛焦顯微鏡觀察,發現經由γ-tocotrienol處理後Ras不活化,綜合上述之結果,γ-tocotrienol使信號蛋白無法異戊二烯化(Isoprenylation),導致Ras不活化,抑制HMG-CoA reductase蛋白表現,進而降低膽固醇生合成之能力。

HMG-CoA reductase is the rate-controlling enzyme of the mevalonate pathway, the metabolic pathway produces cholesterol. γ-Tocotrienol and farnesol has the similar chemical structure and both of them promote the degradation of HMG-CoA reductase. In this study, human hepatoma HepG2 cell line was used to investigate how γ-tocotrienol inactivate HMG-CoA reductase and reduce the biosynthesis of cholesterol. Red yeast rice contains several compounds collectively known as monacolins, substances known to inhibit cholesterol synthesis as well. One of these, monacolin K is a potent inhibitor of HMG-CoA reductase, and is also known as mevinolin or lovastatin. Western blot assay showed that γ-tocotrienol inhibited HMG-CoA reductase protein expression. γ-Tocotrienol and monacolin K were found to decrease total cholesterol in HepG2 cell in a dose dependent manner. γ-Tocotrienol treatment was found to inhibit the translocation of Ras inactivate in HepG2 cell by using confocal microscopy. Taken together, these data suggest that γ-tocotrienol inhibit isoprenylation, and leading to Ras inactivation, subsequently inhibiting HMG-CoA reductase protein expression, and reducing the biosynthesis of cholesterol.

中文摘要....................................................I
Abstract.................................................III
誌謝.......................................................V
目錄......................................................VI
圖表目錄................................................. IX
第一章前言..................................................1
第二章文獻回顧..............................................2
2.1 Gamma-生育三烯醇(γ-tocotrienol)........................2
2.2 莫那可林 K (monacolin K) ...............................5
2.3心血管疾病介紹...........................................6
2.4膽固醇(cholesterol) ....................................8
2.4.1膽固醇的生理功能......................................10
2.5脂蛋白與膽固醇的關係.....................................11
2.6一般常見降膽固醇藥物.....................................13
2.7 Rat sarcoma (Ras)蛋白.................................14
2.8有絲分裂活化蛋白質激酶(mitogen-activated protein kinases pathway, MAPK pathway)....................................16
2.9轉錄因子(nuclear factor κB, NF-κB)......................17
第三章材料與方法............................................19
3.1實驗架構................................................19
3.2藥品試劑................................................20
3.3儀器設備................................................22
3.4細胞試驗................................................24
3.4.1標準品配製............................................24
3.4.2細胞培養藥品配製.......................................24
3.4.2.1胎牛血清............................................24
3.4.2.2 10倍磷酸緩衝液.....................................24
3.4.2.3 1x Trypsin-EDTA之製備.............................24
3.4.3解凍細胞..............................................25
3.4.4冷凍細胞..............................................25
3.4.5細胞繼代培養..........................................25
3.4.6 HepG2細胞株之細胞計數................................26
3.4.7細胞存活率分析........................................26
3.4.8細胞總膽固醇分析......................................28
3.4.9蛋白質電泳與西方點墨法.................................29
3.4.9.1細胞蛋白質萃取......................................29
3.4.9.2蛋白質定量........................................31
3.4.9.3蛋白質電泳分析......................................31
3.4.9.4 SDS電泳...........................................32
3.4.9.5蛋白質轉印..........................................32
3.4.9.6免疫染色法..........................................32
3.5共軛焦顯微鏡觀察........................................33
3.6流式細胞儀分析..........................................34
第四章結果................................................36
4.1 γ-Tocotrienol對於HepG2人類肝癌細胞之存活率測定...........36
4.2 Glucose誘發HepG2人類肝癌細胞之膽固醇增加測定..............36
4.3 γ-Tocotrienol與monacolin K對於HepG2人類肝癌細胞之降低總膽固醇之測定...................................................36
4.4 γ-Tocotrienol對於HepG2人類肝癌細胞之HMG-CoA reductase 表現的影響....................................................37
4.5 Monacolin K對於HepG2人類肝癌細胞之HMG-CoA reductase表現的影響........................................................37
4.6 γ-Tocotrienol對於HepG2人類肝癌細胞中IκBα磷酸化之影響.....38
4.7 γ-Tocotrienol與monacolin K對於HepG2人類肝癌細胞之Ras蛋白轉位現象....................................................38
4.8 γ-Tocotrienol與glucose處理HepG2人類肝癌細胞之NFκB轉位現象........................................................38
4.9 γ-Tocotrienol於HepG2人類肝癌細胞對細胞週期之影響.........39
第五章討論.................................................50
參考文獻...................................................53
作者簡介...................................................59
圖目錄
圖1、民國100年台灣地區國人十大死因............................1
圖2、維生素E的分子立體結構與化學結構...........................2
圖3、Tocotrienol 抑制膽固醇路徑圖............................4
圖4、HMG-CoA和acid form的monacolin K結構....................6
圖5、膽固醇之結構............................................9
圖6、膽固醇體內之代謝........................................9
圖7、脂蛋白的結構...........................................11
圖8、脂質於人體中之代謝.....................................12
圖9、statins抑制膽固醇生合成圖..............................14
圖10、活化的Ras作用於質膜...................................16
圖11、RAS-GTP活化與MAPK pathway............................17
圖12、實驗架構圖...........................................19
圖13、MTT assay ..........................................27
圖14、總膽固醇測定原理圖....................................28
圖15、以濃度2、4、8、16、32 μM之γ-tocotrienol處理細胞24小時,細胞存活之變化...............................................40
圖16、HepG2人類肝癌細胞處理20μM 之glucose於12小時後,誘發之膽固醇增加......................................................41
圖17、以濃度2、4、8、16、32 μM γ-tocotrienol與monacolin K,分別處理24小時,對於HepG2人類肝癌細胞之降低總膽固醇之測定..........42
圖18、以濃度2、4、8、16、32 μM γ-tocotrienol處理24小時,對於HepG2人類肝癌細胞之HMG-CoA reductase 表現的影響..............43
圖19、以濃度2、4、8、16、32 μM monacolin K分別處理24小時,對於HepG2人類肝癌細胞之HMG-CoA reductase 表現的影響............. 44
圖20、γ-Tocotrienol對於HepG2人類肝癌細胞之磷酸化-IκBα表現的影響........................................................45
圖21、γ-Tocotrienol與monacolin K對於HepG2人類肝癌細胞之Ras蛋白轉位現象...................................................46
圖22、γ-Tocotrienol與glucose對於HepG2人類肝癌細胞蛋白轉NFκB位現象........................................................47
圖23、γ-Tocotrienol於HepG2人類肝癌細胞處理24小時,(A) 0、(B) 2 μM、(C) 4 μM、(D) 8 μM、(E) 16 μM、(F) 32 μM,以流式細胞儀偵測Sub-G1之變化,觀察細胞之週期................................48
圖24、γ-Tocotrienol於HepG2人類肝癌細胞處理72小時,A)0、(B) 2 μM、(C)4 μM、(D)8 μM、(E)16 μM、(F)32 μM,以流式細胞儀偵測Sub-G1之變化,觀察細胞之週期....................................49
圖25、γ-Tocotrienol抑制HMG-CoA reductase表現並降低膽固醇生合成之路徑圖....................................................52
表目錄
表1、含有tocotrienols的食物.................................3
表2、冠心病的主要危險因素.....................................7
表3、人類血漿脂蛋白的物理特性與組成(重量百分比)................12
表4、電泳膠體配製...........................................31

張聰明、蕭明裕、彭瓊輝、王文綺 (2003) 生物化學 (初版) 。新文京開發出版有限公司,183~212頁。
蔡樹濤 (2011) 高血脂症 (初版)。元麓書社,21~45頁。
陳嘉芬(2005)細胞生物學(第二版)。藝軒圖書出版社,329~333。
行政院衛生署 (2002) 中華民國公共衛生概況。行政院衛生署編印。
行政院衛生署 (2012) 國人十大死因統計資料。行政院衛生署編印。
花士哲,盧介祥,張天鈞 (2008) 從腫瘤轉移機轉談甲狀腺未分化癌藥物治療之進展。內科學誌,19:472~480。
莊淑慧 (2007) Statins 的多效作用與冠狀動脈疾病。
潘子明 (2005) 真菌保健食品-紅麴製品介紹及國內研究現況。農業生技產業季刊,第三期 28~36頁。
鄧富元、龔素芳、夏毅然、高文斌、謝耀東 (2006) NF-κB在發炎反應、細胞凋亡與癌症生成過程中的調控。中華牙誌,25(1): 12~24。
Acalovschi M (2001) Cholesterol gallstones: from epidemiology to prevention. Postgraduate medical journal. 77(906): 22-1229.
Aggarwal BB, Sundaram C, Prasad S, Kannappan R. (2010) Tocotrienols, the Vitamin E of the 21st Century: It’s Potential Against Cancer and Other Chronic Diseases. Biochem pharmacol. 80(11): 1613-1631.
Barter P. (2005) The role of HDL- cholesterol in preventing atherosclerotic disease. European heart journal supplements. 7: 4-8.
Berkhout TA, Havekes LM, Pearce NJ, Groot PHE. (1990) The effect of (-)-hydroxycitrate on the activity of the low-density-lipoprotein receptor and 3-hydroxy-3-methylglutaryl-CoA reductase levels in the human hepatoma cell line HepG2. Biochemical journal. 272(1): 181-218.
Brown MS, Kovanen PT, and Gordon JL. (1981) Regulation of plasma cholesterol by lipoprotein receptors. Science . 212(4495): 628-635.
Clayton PT. (1998) Disorders of cholesterol biosynthesis. Archives of disease in childhood. 78(2): 185-189.
DeBose-Boyd RA. (2008) Feedback regulation of cholesterol synthesis: sterol- accelerated ubiquitination and degradation of HMG CoA reductase. Cell research. 18(6): 609- 621.
Downward J. (2003) Targeting RAS signalling pathways in cancer therapy. Nature reviews cancer. 3(1): 11-22.
Endo A. (1992) The discovery and development of HMG-CoA reductase inhibitors. Journal of lipid research. 53: 1569-1582.
Edwards PA, Cornell CC and Ng L. (1994) Identification of farnesol as the non-sterol derivative of mevalonic acid required for the accelerated degradation of 3-Hydroxy-3-methylglutarylcoenzyme A reductase. The journal of biological chemistry. 269(26): 17390-17393.
Feo F, Frau M, Tomasi ML, Brozzetti S, Pascale RM. (2009) Genetic and epigenetic control of molecular alterations in hepatocellular carcinoma. Experimental biology and medicine. 234(7): 726-736.
Francis GA, Johnson RL, Findlay JM, Wang J, Hegele RA. (2005) Cerebral cholesterol granuloma in homozygous familial hyper-
cholesterolemia. Canadian medical association or its licensors. 172 (4): 495-497.
Frank A, Chin XWD, Schrader C, Eckert Gp, Rimbach G. (2012) Do tocotrienols have potential as neuroprotective dietary factors? Ageing research reviews. 11(1): 163-180.
Goldberg AC, and Schonfeeld G. (1985) Effer of diet on lipoprotein metabolism. Annual review of nutrition. 5: 195-212.
Hall G, Rogers TB and Hasday JD. (2006) Regulating the regulator: NF-κB signaling in heart. Journal of molecular and cellular cardiology. 41(4): 580–591.
Heber D, Yip I, Ashley JM, Elashoff DA, Elashoff RM, Go VLW. (1999) Cholesterol-lowering effects of a proprietary Chinese red-yeast-rice dietary supplement. American society for clinical nutrition. 69(2): 231–236.
Graaf MR, Richel DJ, Noorden CJ, Guchelaar HJ. (2004) Effects of statins and farnesyltransferase inhibitors on the development and progression of cancer. Cancer treatment reviews. 30(7): 609-641.
Ikeda S, Tohyama T, Yoshimura H, Hamamura K, Abe K, Yamashita K (2003). Dietary alpha-tocopherol decreases alpha-tocotrienol but not gamma-tocotrienol concentration in rats. American society for nutritional sciences. 133(2): 428-434.
Ju1 J, Picinich SC, Yang Z, Zhao Y, Suh1 N, Kong AN, Yang CS. (2010) Cancer-preventive activities of tocopherols and tocotrienols. Carcinogenesis. 31(4): 533-542.
Kuhlmann J and Pechlivanis M. (2006) Hydrophobic modifications of Ras proteins by isoprenoid groups and fatty acids-More than just membrane anchoring. Biochimica et biophysica acta. 1764(12): 1914- 1931.
McAnally JA, Jung M, Mo H. (2003) Farnesyl-O-acetylhydroquinone and geranyl-O-acetylhydroquinone suppress the proliferation of murine B16 melanoma cells, human prostate and colon adenocarcinoma cells, human lung carcinoma cells, and human leukemia cells. Cancer letters. 202(2): 181-192.
McCubrey JA, Abrams SL, Steelman LS, Lee JT, Chan F, Bertran FE, Navolanic PM, Franklin RA, Terrian DM, D’Assoro AB, Salisbury JL, Mazzarino MC, Stivala F, Libra M. (2006) Roles of the RAF/MEK /ERK and PI3K/PTEN/AKT pathways in malignant transformation and drug resistance. Advances in biological regulation. 46: 249–279.
Meigs TE, Roseman DS and Simoni RD. (1996) Regulation of 3-Hydroxy-3-methylglutaryl-coenzyme A reductase degradation by the nonsterol mevalonate metabolite farnesol in vivo. The journal of biological chemistry. 271(14): 7916-7922.
Mayes PA, Murray R K, Granne D K, Rodwell VW. (1988) Haprper’s biochemistry. (21st Edition). 241-252, 226-240.
Menge T, Hartung HP and Stuve O. (2005) Statins-a cure-all for the brain? Nature reviews neuroscience. 6(4): 325-331.
Meigs TE and Simoni RD. (1997) Farnesol as a regulator of HMG-CoA reductase degradation: characterization and role of farnesyl pyrophosphatase. Archives of biochemistry and biophysics. 345(1): 1-9.
Li T, Chanda D, Zhang Y, Choi HS and Chiang JYL. (2010) Glucose stimulates cholesterol 7α-hydroxylase gene transcription in human hepatocytes. The journal of lipid research. 51(4): 832-842.
Overmeyer JH and Maltese WA. (2011) Death pathways triggered by activeated Ras in cancer cells. Frontiers in bioscience. 16: 1693-1713.
Ness GC, Chambers C, Zhao Z, Keller RK. (1996) Farnesol is not the nonsterol regulator mediating degradation of HMG-CoA reductase in rat liver. Archives of biochemistry and biophysics. 328(2): 324–330.
Nimal Ratnayake W N and Gilani GS. (2004) Nutrition and health effects of dietary fats. Pakistan journal of nutrition. 3(4): 205-212.
Rodwell VW and Friesen JA. (2004) The 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductases. Genome biology. 5: 248-254.
Roy S, Khanna S and Sen CK. (2006) Tocotrienols: vitamin E beyond tocopherols. Life sciences. 78(18): 2088-2098.
Roy M, Kung HJ, Ghosh PM. (2011) Statins and prostate cancer: role of cholesterol inhibition vs. prevention of small GTP-binding proteins. American journal of cancer research. 1(4): 542-561.
Srivastava JK and Gupta S. (2006) Tocotrienol-rich fraction of palm oil induces cell cycle arrest and apoptosis selectively in human prostate cancer cells. Biochemical and biophysical research communications. 346(2): 447-453.
Su YC, Wang JJ, Lin TT, Pan TM. (2003) Production of the secondary metabolites c-aminobutyric acid and monacolin K by Monascus. The journal of industrial microbiology and biotechnology. 30(1): 41-46.
Sylvester PW. (2012) Synergistic anticancer effects of combined γ- tocotrienol with statinor receptor tyrosine kinase inhibitor treatment. Genes and nutrition. 7(1): 63-74.
Tan B. (2005) Appropriate spectrum vitamin E and new perspectives on desmethyl tocopherols and tocotrienols. The journal of the american neutricutical association. 8(1): 152-4524.
Theriault A, Chao JT, Wang Q, Gapor A, Adeli K. (1999) Tocotrienol: a review of its therapeutic potential. Clinical biochemistry. 32(5): 309-319.
Theriault A, Wang Q, Gapor A, Adeli K. (1999) Effects of γ-Tocotrienol on apoB synthesis. degradation, and secretion in HepG2 cells. Arteriosclerosis, thrombosis, and vascular biology. 19(3): 704-712.
Thomas X and Elhamri M. (2007) Tipifarnib in the treatment of acute myeloid leukemia. Biologics. 1(4): 415-424.
Vieraanthony J, Mph MD, Sheridan S. (2010) Global Risk of Coronary Heart Disease: Assessment and Application. American family physician. 82(3): 265-274.
Yap WN, Chan PN, Han HY, Lee DTW, Ling MT, Wong YC, Yap YL. (2008) γ-Tocotrienol suppresses prostate cancer cell proliferation and invasion through multiple-signalling pathways. British journal of cancer. 99(11): 1832-1841.

連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
1. 張國峻、李文昭(2010)酚/間苯二酚共溶劑液化之酚化柳杉木材之性質。林業研究季刊32(1):77-86。
2. 張上鎮、吳季玲、王升陽(1997)熱分析技術在木材化學研究領域之應用。林產工業16(1):133-148。
3. 林曉洪、王秀華、彭嘉文(1997)9 mm耐燃合板製造之研究。中華林學季刊30(4):437-444。
4. 林曉洪、王秀華、陳川助(1991)防火劑表面刷塗處理四種商用木材之抗燃性及吸脫濕性研究。林產工業10(2):45-65。
5. 林曉洪、王松永(1988)防火劑浸漬處理對造林木材抗燃性之改善效應。林產工業7(3):1-12。
6. 林曉洪、王秀華(1999c)混合型耐燃劑之抑焰性與耐燃性效應。林產工業 18(4):427-442。
7. 林曉洪、王秀華(1999b)氧氣指數評估耐燃處理材之效果。林產工業18(2):195-202。
8. 林曉洪、王秀華(1999a)不同藥劑處理對台灣杉木材熱重分析之影響。林產工業18 (3) :257-268。
9. 林曉洪、王秀華(1998)耐燃處理合板之耐燃性改善效應。林產工業17(1):89-102。
10. 林曉洪、王秀華(1989a)防火劑浸漬處理對壁紙及窗簾紙抗燃性之改善效應林產工業8(3):33~44。
11. 林曉洪(2006)氧氣指數評估阻燃藥劑雙重處理放射松單板之抑焰效應。中華林學季刊 39(1):81-92。
12. 鄒哲宗、林佳佳、林喬屏(2004)磷酸胍熱浸處理紅柳桉單板之熱重量分析。中華林學季刊37(4):427-434。
13. 花士哲,盧介祥,張天鈞 (2008) 從腫瘤轉移機轉談甲狀腺未分化癌藥物治療之進展。內科學誌,19:472~480。