跳到主要內容

臺灣博碩士論文加值系統

(44.201.99.222) 您好!臺灣時間:2022/12/04 00:18
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:楊程茗
研究生(外文):Chang-Ming Yang
論文名稱:內皮素-1調節膽固醇恆定之角色
論文名稱(外文):The Regulatory Mechanism of Endothelin-1 on Cholesterol Homeostasis in Hepatocyte
指導教授:阮琪昌
指導教授(外文):Chi-Chang Juan
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生理學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:104
語文別:中文
論文頁數:53
中文關鍵詞:內皮素-1
外文關鍵詞:Endothelin-1
相關次數:
  • 被引用被引用:0
  • 點閱點閱:223
  • 評分評分:
  • 下載下載:4
  • 收藏至我的研究室書目清單書目收藏:0
內皮素為一種強大的血管收縮素,經由大多數的內皮細胞製造且分泌。先前的研究顯示內皮素會影響胰島素訊息傳導和後續的葡萄糖攝取的能力;此外我們也發現內皮素會刺激脂肪細胞的脂肪分解,可知內皮素-1具有調節脂肪相關代謝之能力。但是目前尚不清楚內皮素-1對於肝臟脂肪代謝之影響。反向膽固醇運送為肝臟細胞將身體過多的膽固醇排出體外之機制。反向膽固醇運輸為一個複雜的機制,包括將周邊組織細胞內的膽固醇流出至高密度脂蛋白之中,之後高密度脂蛋白經過血液循環之運送至肝臟,而高密度脂蛋白之中的膽固醇和膽固醇酯流入肝臟細胞,之後肝臟細胞則將流入的膽固醇進一步的代謝與合成膽汁排出體外。然而肝臟中的清道夫接受器B第一型(SR-B1)、ATP結合盒轉運器A第一型(ABCA1)、低密度脂蛋白接受器相關蛋白1(LRP1)和低密度脂蛋白接受體(LDLR)在RCT循環中扮演重要的角色。本實驗的研究目的為探索ET-1對於肝細胞RCT調節機制,首先我們利用Western blot來偵測ET-1是否影響ABCA1、LDLR、LRP1、SR-BI之蛋白質表現,,而ET-1影響膽固醇流出和攝取利用TopFluor-Cholesterol和DiI-HDL來測定。結果顯示ET-1在於HepG2中,有劑量依賴性的降低ABCA1蛋白質表現量,但是在於LDLR、LRP1和SR-B1的蛋白質表現量沒有改變,且ET-1處理顯著抑制膽固醇流出。進一步共同處理ET-1的專一性接受器阻斷劑內皮素A型接受器阻斷劑 (BQ610) 或內皮素B型接受器阻斷劑(BQ788),結果顯示BQ788與ET-1共同處理的HepG2之中,受到ET-1所減少的ABCA1蛋白質表現量和膽固醇流出率有回復之現象。進一步利用PI3K阻斷劑(LY294002)、ERK阻斷劑 (PD98059)、 JNK inhibitor (SP600125)、p38 MAPK阻斷劑(SB203580)或PKC阻斷劑(Ro318220)探討其訊息路徑,結果顯示前處理 LY294002的HepG2之中,受到ET-1調節減少的ABCA1蛋白質表現量有回復之現象,且在於前處理LY294002的HepG2之中,受到ET-1調節減少的膽固醇流出率也有回復之現象。進一步利用cycloheximide抑制蛋白質轉錄抑制劑來探討ET-1造成ABCA1之減少之機轉,結果顯示在HepG2中,處理ET-1促進ABCA1蛋白質分解。根據以上結果,證實ET-1與ETBR結合活化PI3K訊息路徑,促進ABCA1蛋白質分解,進而使膽固醇流出率下降。
Endothelin-1 (ET-1), a powerful vasoconstricting polypeptide, primarily produced and secreted by vascular endothelial cells. Our studies have demonstrated that ET-1 impaired insulin signal pathway and subsequent glucose uptake in vitro and in vivo. Besides, we also demonstrated that ET-1 stimulated lipolysis in adipocytes. These findings suggested that ET-1 could regulate lipid-associated metabolism. However, the regulation of ET-1 on hepatic lipid metabolism is still not clear. Reverse cholesterol transport (RCT) is a mechanism regulating the removal of excess body cholesterol through hepatocytes. RCT is a complex mechanism including the efflux of cellular cholesterol to high density lipoprotein (HDL) from peripheral cells, the transport of lipoprotein-cholesterol in blood to the liver, and the delivery of cholesterol esters to hepatocytes from HDL for its metabolism and biliary excretion. Hepatic scavenger receptor type B class I (SR-BI), ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), low density lipoprotein receptor related protein 1 (LRP1) and low density lipoprotein receptor (LDLR) play important roles in the RCT. The purpose of this study is to explore the regulatory mechanism of ET-1 on RCT in HepG2 cells. We first investigated the effect of ET-1 on expression of SR-BI, LDLR, LRP1, ABCA1 by western blot. Effects of ET-1 on cholesterol efflux and cholesterol uptake were also determined by using TOPFLOUR-cholesterol and DiI-HDL. To further clarify the mechanism mediating ET-1 actions on RCT, ET-1 receptor antagonists or specific signal inhibitors were administrated and ET-1-regulated expression of SR-BI, LDLR, LRP1, ABCA1, and cholesterol efflux and cholesterol uptake were measured. Our results showed that ET-1 significantly decreased ABCA1 protein expression and cholesterol effux in HepG2 cells. However, ET-1 has no effect on SR-B1, LDLR, LRP1 protein expression. Pretreatment of ET type B receptor (ETBR) blocker BQ788, but not ETAR blocker BQ610, successfully prevented ET-1-suppressed ABCA1 expression and cholesterol efflux. In addition, pretreatment of PI3K inhibitor (LY294002), but not ERK inhibitor (PD98059), JNK inhibitor (SP600125) or PKC inhibitor (Ro318220), or p38MAPK inhibitor (SB203580), prevented ET-1-suppressed ABCA1 expression. Moreover, pretreatment of LY294002 also blocked ET-1-reduced cholesterol efflux. Besides, cells were pretreated with protein synthesis inhibitors cycloheximide and effect of ET-1 and ABCA1 was evaluated. The result showed that ET-1 significantly decreased ABCA1 protein levels and suggested that ET-1 could decrease ABCA1 protein stability. In conclusion, ET-1, acting via the ETBR, decreased ABCA1 protein expression and cholesterol efflux through PI3K-depended pathway.
目錄 i
中英文名詞與縮寫 iv
中文摘要 v
Abstract vii
第一章、 前言 1
第二章、 文獻回顧 2
一、 内皮素 2
1. 內皮素簡單介紹 2
2. 內皮素-1的生合成與分解 3
3. 內皮素-1與脂質代謝 3
二、肝臟代謝 4
1.肝臟組織與生理功能 4
2. 肝臟脂質代謝與運送 6
3. 反向膽固醇運送 7
第三章、材料與方法 10
一、實驗設計 10
二、藥品抗體來源 15
三、細胞培養 16
四、西方墨點法 18
五、膽固醇流出 21
六、膽固醇攝取 21
七、肝臟脂質萃取 22
八、三酸甘油酯含量測定 22
九、總膽固醇含量測定 22
十、動物實驗 23
十一、統計方法 23
第四章、 實驗結果 25
(1)ET-1對於HepG2肝臟細胞膽固醇運送相關蛋白之劑量效應 25
(3)ET-1對於HepG2肝臟細胞的膽固醇流出之影響 26
(4)ET-1對於HepG2肝臟細胞的膽固醇攝取之影響 26
(5)ET-1接受器對於HepG2肝臟細胞中膽固醇運送相關蛋白之影響 26
(6)ET-1接受器對於HepG2肝臟細胞膽固醇流出之影響 27
(7)ET-1影響膽固醇運送相關蛋白之訊息路徑 28
(8)ET-1影響 HepG2肝臟細胞膽固醇流出之訊息路徑 28
(9)ET-1對於HepG2肝臟細胞ABCA1蛋白質穩定性之影響 29
(10)ET-1對於大鼠肝臟脂質代謝之影響 29
第五章、討論 31
第六章、結論 34
參考文獻 35
圖表與說明 40
附表一 53


1. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988; 332:411-415
2. Yanagisawa M, Inoue A, Ishikawa T, Kawuya Y, Kimura S, Kumagaye S, Nakajima K, TX W, Sakakibara S, Goto K. Primary structure, synthesis, and biological activity of rat endothelin, an endothelium-derived vasoconstrictor peptide. Proc Natl Acad Sci U S A 1988; 85:6964-6967.
3. Saida K, Mitsui Y, Ishida N. A novel peptide, vasoactive intestinal contractor, of a new (endothelin) peptide family: molecular cloning, expression, and biological activity. J Biol Chem 1989; 264:14613-14616.
4. Zhang Y, Abdel-Latif AA. Activation of phospholipase D by endothelin-1 and other pharmacological agents in rabbit iris sphincter smooth muscle cell. Cell Signal 1992; 4
5. Wagner OF, Christ G, Wojta J, Vierhapper H, Parzer S, Nowotny PJ, Schneider B, Waldhäusl W, Binder BR. Polar secretion of endothelin-1 by cultured endothelial cells. J Biol Chem 1992; 267:16066-16068.
6. Miyauchi T, Yanagisawa M, Iida K, Ajisaka R, Suzuki N, Fujino M, Goto K, Masaki T, Sugishita Y. Age- and sex-related variation of plasma endothelin-1 concentration in normal and hypertensive subjects. Am Heart J 1992; 123:1092-1093.
7. Xu D EN, Giaid A. ECE-1: a membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothelin-1. Cell 1994; 78:473-485.
8. Inoue A, Yanagisawa M, Takuwa Y, Mitsui Y, Kobayashi M, Masaki T. The human preproendothelin-1 gene. J Biol Chem 1989; 264:14954-14959.
9. Yanagisawa M, Masaki T. Molecular biology and biochemistry of the endothelins. Trends Pharmacol Sci 1989; 10:374-378.
10. de Nucci G, Thomas R, D’Orleans-Juste P, Antunes E, Walder C, Warner TD, JR V. Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc Natl Acad Sci U S A 1998; 85:9797-9800.
11. Clozel M, Hess P, Rey M, Iglarz M, Binkert C, Qiu C. Bosentan, sildenafil, and their combination in the monocrotaline model of pulmonary hypertension in rats. Exp Biol Med (Maywood) 2006; 231:967-973.
12. Davar G, Hans G, Fareed MU, Sinnott C, Strichartz G. Behavioral signs of acute pain produced by application of endothelin-1 to rat sciatic nerve. Neuroreport 1998; 9::2279-2283.
13. Ju C, Ye M, Li F. Plasma Brain Natriuretic Peptide, Endothelin-1, and Matrix Metalloproteinase 9 Expression and Significance in Type 2 Diabetes Mellitus Patients with Ischemic Heart Disease. Med Sci Monit 2015; 21:2094-2099.
14. Li C1, Li J, Weng X, Lan X, Chi X. Farnesoid X receptor agonist CDCA reduces blood pressure and regulates vascular tone in spontaneously hypertensive rats. J Am Soc Hypertens 2015; 9:507-516.
15. Schiffrin EL. Endothelin: potential role in hypertension and vascular hypertrophy. Hypertension 1995; 25:1135-1143.
16. Hu Z, Zhang J, Guan A, Gong H, Yang M, Zhang G, Jia J MH, Yang C, Ge J ZY. Granulocyte colony-stimulating factor promotes atherosclerosis in high-fat diet rabbits. Int J Mol Sci 2013; 4:4805-4816.
17. Lteif A, Vaishnava P, Baron AD, Mather KJ. Endothelin limits insulin action in obese/insulin-resistant humans. Diabetes 2007; 56:728-734
18. Mangiafico RA, Malatino LS, Santonocito M, Spada RS. Plasma endothelin-1 concentrations in non-insulin-dependent diabetes mellitus and nondiabetic patients with chronic arterial obstructive disease of the lower limbs. Int Angiol 1998; 17:97-102.
19. Juan CC, Chien Y, Wu LY, Yang WM, Chang CL, Lai YH, Ho PH, Kwok CF, Ho LT. Angiotensin II enhances insulin sensitivity in vitro and in vivo. Endocrinology 2006; 146:2246-2254
20. Juan CC, Chang LW, Huang SW, Chang CL, Lee CY, Chien Y, Hsu YP, Ho PH, Chen YC, Ho LT. Effect of endothelin-1 on lipolysis in rat adipocytes. Obesity (Silver Spring) 2006; 14:398-404.
21. Tran-Thi TA, Kawada N, Decker K. Regulation of endothelin-1 action on the perfused rat liver. FEBS Lett 1993; 318:353-357.
22. Wereszczynka-Siemiatkowska U, Swidnicka-Siergiejko A, Siemiatkowski A, Bondyra Z, Wasielica-Berger J, Mroczko B, Janica J, Dabrowski A. Endothelin 1 and transforming growth factor-β1 correlate with liver function and portal pressure in cirrhotic patients. Cytokine 2015; [Epub ahead of print].
23. Theodorakis N, Maluccio M, Skill N. Murine study of portal hypertension associated endothelin-1 hypo-response. World J Gastroenterol 2015.; 21:4817-4828
24. Lin CY, Lee TS, Chen CC, Chang CA, Lin YJ, Hsu YP, Ho LT. Endothelin-1 exacerbates lipid accumulation by increasing the protein degradation of the ATP-binding cassette transporter G1 in macrophages. J Cell Physiol 2011; 226:2198-2205.
25. Abdel-Misih SR, Bloomston M. Liver anatomy. Surg Clin North Am 2010; 90:643-653.
26. Abdel-Misih SR1, Bloomston M. Liver anatomy. Surg Clin North Am 2010; 90:643-653.
27. Bismuth H. Surgical anatomy and anatomical surgery of the liver. World J Surg 1982; 6:3-9.
28. Oosterveer DM, Versmissen J, Yazdanpanah M, Defesche JC, Kastelein JJ, Sijbrands EJ. The risk of tendon xanthomas in familial hypercholesterolaemia is influenced by variation in genes of the reverse cholesterol transport pathway and the low-density lipoprotein oxidation pathway. Eur Heart J 2010; 31:1007-1012.
29. Sharrett AR, Ballantyne CM, Coady SA, Heiss G, Sorlie PD, D C. Patsch WCoronary heart disease prediction from lipoprotein cholesterol levels, triglycerides, lipoprotein(a), apolipoproteins A-I and B, and HDL density subfractions: The Atherosclerosis Risk in Communities (ARIC) Study. Circulation 2001; 104:1108-1113.
30. Olsson AG, Schwartz GG, Szarek M, Sasiela WJ, Ezekowitz MD, Ganz P, Oliver MF, Waters D, Zeiher A. High-density lipoprotein, but not low-density lipoprotein cholesterol levels influence short-term prognosis after acute coronary syndrome: results from the MIRACL trial. Eur Heart J 2005; 26:890-896.
31. Angelovich TA, Hearps AC, Jaworowski A. Inflammation-induced foam cell formation in chronic inflammatory disease. Immunol Cell Biol 2015; [Epub ahead of print].
32. Navab M, Reddy ST, Van Lenten BJ, AM F. HDL and cardiovascular disease: atherogenic and atheroprotective mechanisms. Nat Rev Cardiol 2011; 8:222-232.
33. Rohatgi A. High-Density Lipoprotein Function Measurement in Human Studies: Focus on Cholesterol Efflux Capacity. Prog Cardiovasc Dis 2015; 58:32-40.
34. Oh GS, Yoon J, Lee GG, Oh WK, Kim SW. 20(S)-protopanaxatriol inhibits liver X receptor α-mediated expression of lipogenic genes in hepatocytes. J Pharmacol Sci 2015; 128:71-77.
35. Venkateswaran A, Laffitte BA, Joseph SB, Mak PA, Wilpitz DC, Edwards PA, Tontonoz P. Control of cellular cholesterol efflux by the nuclear oxysterol receptor LXR alpha. Proc Natl Acad Sci U S A 2000; 97:12097-12102.
36. Ferré P, Foufelle F. Hepatic steatosis: a role for de novo lipogenesis and the transcription factor SREBP-1c. Diabetes Obes Metab 2010; 12:83-92.
37. Postic C, Girard J. Contribution of de novo fatty acid synthesis to hepatic steatosis and insulin resistance: lessons from genetically engineered mice. J Clin Invest 2008; 118:829-838.
38. Oh GS, Lee GG, Yoon J, Oh WK, Kim SW. Selective inhibition of liver X receptor α-mediated lipogenesis in primary hepatocytes by licochalcone. Chin Med 2015; 10:1-8.
39. FOLCH J, LEES M, SLOANE STANLEY GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 1957; 226:497-509.
40. Mizuno T, Hayashi H, Kusuhara H. Cellular Cholesterol Accumulation Facilitates Ubiquitination and Lysosomal Degradation of Cell Surface-Resident ABCA1. Arterioscler Thromb Vasc Biol 2015; 35:1347-1356.
41. Rust S, Rosier M, Funke H, Real J, Amoura Z, Piette JC, Deleuze JF, Brewer HB, Duverger N, Denèfle P, Assmann G. Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1. Nat Genet 1999; 22:352-355.
42. Parks JS, Chung S, Shelness GS. Hepatic ABC transporters and triglyceride metabolism. Curr Opin Lipidol 2012; 23:196-200.
43. Gandhi CR, Behal RH, Harvey SAK, Nouchi, T.A, Olson, M.S. Hepatic effects of endothelin: Receptor characterization and endothelin-induced signal transduction in hepatocytes. 1992; 287:897-904.
44. Nie S, Zhou J, Bai F, Jiang B, Chen J, Zhou J. Role of endothelin A receptor in colon cancer metastasis: in vitro and in vivo evidence. Mol Carcinog 2014; 53:85-91.
45. Rodriguez MR, Soria LR, Ventimiglia MS, Najenson AC, Di María A, Dabas P, Fellet A, Marinelli RA, Vatta MS, Bianciotti LG. Endothelin-1 and -3 induce choleresis in the rat through ETB receptors coupled to nitric oxide and vagovagal reflexes. Clin Sci (Lond) 2013; 125:521-532.
46. Lim-Kyu Lee, Ju-Hyun Kim, Mee-Young Kim, Jeong-Uk Lee, Seung-Min Yang, Hye-Joo Jeon, Won-Deok Lee, Woong Noh, Taek-Yong Kwak, Sung-Ho Jang, Tae-Hyun Lee, Bokyung Kim D, Junghwan Kim P. A Review of Signal Transduction of Endothelin-1 and Mitogen-activated Protein Kinase-related Pain for Nanophysiotherapy. J Phys Ther Sci 2014; 26:789–792.
47. Kim B, Kim J, Bae YM, Cho SI, Kwon SC, Jung JY, Park JC, Ahn HY. p38 mitogen-activated protein kinase contributes to the diminished aortic contraction by endothelin-1 in DOCA-salt hypertensive rats. Hypertension 2004; 43:1086-1091
48. Chai SP, Fong JC. Synergistic induction of insulin resistance by endothelin-1 and cAMP in 3T3-L1 adipocytes. Biochim Biophys Acta 2015; [Epub ahead of print].
49. Juan CC, Chang CL, Lai YH, Ho LT. Endothelin-1 induces lipolysis in 3T3-L1 adipocytes. Am J Physiol Endocrinol Metab 2005; 288:1146-1152.
50. Cain AE, Tanner DM, Khalil RA. Endothelin-1--induced enhancement of coronary smooth muscle contraction via MAPK-dependent and MAPK-independent [Ca(2+)](i) sensitization pathways. Hypertension 2002; 39:543-549.


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