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研究生:李育憲
研究生(外文):Yu-Hsien Li
論文名稱:在體外模式下利用週期性機械力探討心肌細胞內加壓素基因表現及其分子調控之機轉
論文名稱(外文):The molecular regulation and the gene expression of urotemsin II by cyclic stretch in cultured rat cardiomyocytes
指導教授:林建煌林建煌引用關係
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學研究所
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:2008
畢業學年度:97
語文別:中文
論文頁數:68
中文關鍵詞:加壓素
外文關鍵詞:urotensin II
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加壓素是一種血管收縮促進因子,可能參與高血壓和冠狀動脈硬化症的形成。目前研究顯示,在體外實驗模式下,加壓素可作為心肌細胞肥大的指標基因,並可能與心血管疾病上扮演特定連結。在臨床上已有證據顯示,加壓素在心血管疾病上可視為一種獨立潛在危險因子,在心肌細胞肥大及鬱血性心衰竭時,血漿中的加壓素含量會顯著增加。也有一些相關的研究指出在心血管系統中,ROS 也可能刺激加壓素的增加,進而誘發心肌細胞肥大和心肌纖維化,以致可以加速心血管疾病的病程變化,但目前對其分子調節機轉並不清楚。機械力過度負荷是造成心臟疾病原因之一,利用直接的機械展延來研究加壓素在心肌細胞的表現尚未被探討過,而且我們認為加壓素在機械展延下之表現可能與 ROS 的調節作用有關。因此本篇研究的目的:在體外模式下利用週期性機械力探討心肌細胞內加壓素基因表現及其分子調控之機轉。本研究是將心肌細胞接種於一矽膠彈性膜上,再配合機械展延儀器,於每分鐘 60次循環下達到 20% 或 10% 的展延程度。結果發現週期性機械力明顯地會使加壓素蛋白質與其訊息核糖核酸分子表現上升也會透過 STAT1 使的去氧糖核酸-蛋白質轉錄因子的結合活性能力上升。當加入相關抑制劑 ( PD98059, NAC, ERK siRNA, urotensin II siRNA ) 後則又會使的加壓素蛋白質和去氧核糖核酸-蛋白質轉錄因子的結合活性表現下降。最後得到的結論則是:心肌細胞在週期性機械力作用後會經由 ERK 和 STAT1 訊息傳遞路徑影響加壓素上升最後誘發導致心肌纖維化等等的相關心血管疾病。
Urotensin II is a potent vasoconstrictive peptide and involves in hypertension and atherosclerosis. A recent study identified that cardiomyocytes are able to express urotensin II receptor and receive the direct hypertroplic signal from urotensin II. Clinical evidence implicated that urotensin II as a potential independent risk factor for coronary heart disease which increased plasma urotensin II levels are correlated with cardiac hypertrophy and congestive heart failure. Thus, it suggested that urotensin II may play an important role in biological link of cardiovascular pathophysiologies. Several other studies have also indicated that ROS is also able to stimulate the generation of urotensin II in cardiovascular system and leading to cardiac hypertrophy. While urotensin II has been shown increase the degree of cardiovascular diseases but its molecular regulation mechanism remains unclear. Mechanical force overload is both cause and consequences of most heart diseases. How cyclical mechanical stretch affects the regulation of urotensin II in cardiacmyocytes has not been previously characterized. We sought to investigate the cellular and molecular mechanisms of regulation of urotensin II by cyclic stretch in cadiacmyocytes. In the present study, cadiacmyocytes grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation, at 60 cycles/min. Cyclic stretch significantly increased urotensin II protein and mRNA expression after stretch. Cyclic stretch also significantly increased DNA-protein binding activity of STAT1. Addition of PD98059, NAC, ERK siRNA, urotensin II siRNA 30 minutes before stretch inhibited the induction of urotensin II protein and abolished the DNA-protein binding activity induced by cyclic stretch. In conclusion, cyclic mechanical stretch enhances urotensin II expression in cultured cadiacmyocytes. The stretch-induced urotensin II is mediated by angiotensin II and ROS, at least in part, through ERK and STAT1 pathway.
目錄
中文摘要 ……………………………………………………………1
英文摘要 ……………………………………………………………3
一、研究背景 ………………………………………………………5
動脈粥樣硬化 ( Atherosclerosis ) ……………………5
Urotensin II …………………………………………………7
機械性展延(Mechanical Stretch) ……………………8
心臟肥大(Cardiac Hypertrophy) …………………10
二、研究動機 …………………………………………………………13
三、研究方法與步驟…………………………………………………14
心肌細胞分離與培養
( Primary cardiomycytes culture ).14
心肌細胞周期性展延
(In vitro cyclical stretch on culturedcardiomycytes)15
心肌細胞核醣核酸的抽取 ( RNA extraction ……………16
去氧核糖核酸探針之製備( cDNA probe preparation )…17
- 反轉錄 ( Reverse transcription ) ……………17
- Real-time quantitative PCR ……………………17
- 純化PCR 後產物 ( PCR product purification ).18
Total protein 的抽取 ( Total protein extraction ) .19
西方墨點轉印法 ( Western Blotting )…………………20
RNA 干擾 ( RNAinterference )……………………………22
促進子活性分析法 ( Promoter acivity assay )………22
Angiotensin II 酵素免疫分析法
( Enzyme-linked immunoassay , ELISA ) …………………23
免疫螢光染色分析法
( Immunofluorescence staining assay )…………………24
電泳膠移動分析
( Electrophoretic Mobility ShiftAssay )…………… 25
- 核蛋白的抽取 …………………………………………25
- Oligomer DNA primer preparation ………………26
- End labeling probe …………………………………27
- Gel shifting …………………………27
統計分析…………………………………28
四、結果 ……………………………………………………………30
Urotensin II 蛋白質表現 ……………………………30
Urotensin II mRNA 表現 ……………………………30
ERK1/2 (p42/p44 MAP kinase)的活化 ……………31
抑制劑與RNA干擾 …………………………………………33
促進子活性 ………………………………………………34
Gel Shifting 表現 …………………………………………35
五、討論 …………………………………………………………38
六、參考文獻 ……………………………………………………43
圖表 …………………………………………………………………50
Samuel JL, Vandenburgh HH. Mechanically induced orientation of adult
rat cardiac myocytes in vitro. In Vitro Cell Dev Biol. 1990 ; 26:905-914.

Terracio L, Miller B, Borg TK. Effects of cyclic mechanical stimulation
of the cellular components of the heart: in vitro. In Vitro Cell Dev Biol.1988 ; 24:53-58.

Yamazaki T, Komuro I, Yazaki Y. Signalling pathways for cardiac
hypertrophy. Cell Signal. 1998 ; 10:693-698.

Wang BW, Chang H, Lin S, Kuan P, Shyu KG. Induction of matrix
metalloproteinases-14 and 2 by cyclical mechanical stretch is mediated by tumor necrosis factor-a in cultured human umbilical vein endothelial cells. Cardiovasc Res. 2003 ; 59:460-469.

Krum H, Kemp W, et al. Therapeutic potential of blockade of the
urotensin II system in systemic hypertension. Curr Hypertens Rep. 2007 Mar ; 9(1):53-8.

Shyu KG, Wang BW, Yang YH, Tsai SC, Lin S, Lee CC. Amphetamine
activates connexin43 gene expression in cultured neonatal rat cardiomyocytes through JNK and AP-1 pathway. Cardiovasc Res. 2004 ; 63:98-108.
Shyu KG, Chen JJ, Shih NL, Wang DL, Chang H, Lien WP, Liew CC.
Regulation of human cardiac myosin heavy chain genes by cyclical
mechanical stretch in cultured cardiocytes. Biochem Biophys Res
Commun. 1995;210:567-573.

Kemp W, Roberts S, Krum H, et al. Urotensin II: a vascular mediator in
health and disease. Curr Vasc Pharmacol. 2005 Apr ; 3(2):159-68.

Rajapurohitam V, Gan XT, Kirshenbaum LA, Karmazyn M.
The obesity-associated peptide leptin induces hypertrophy in neonatal rat ventricular myocytes. Cir Res. 2003 Aug ; 93:277– 279.

Mat susaka S, Wakabayashi I. Enhancement of vascular smooth muscle
cell migration by urotensin II. Naunyn Schmiedebergs Arch Pharmacol. 2006 Aug ; 373(5):381-6.

Suguro T, Watanabe T, Ban Y, Kodate S, Misaki A, Hirano T, Miyazaki
A, Adachi M. Increased human urotensin II levels are correlated with carotid atherosclerosis in essential hypertension. Am J Hypertens. 2007 Feb;20(2):211-7.

Watanabe T, Itokawa M, Nakagawa Y, Iguchi T, Katagiri T. Increased
levels of insulin-like growth factor binding protein-3 in hypertensive patients with carotid atherosclerosis. Am J Hypertens. 2003 Sep ; 16(9 Pt 1):754-60.

Djordjevic T, BelAiba RS, Bonello S, Pfeilschifter J, Hess J, Görlach
A. Human urotensin II is a novel activator of NADPH oxidase in human pulmonary artery smooth muscle cells. Arterioscler Thromb Vasc Biol. 2005 Mar ; 25(3):519-25.

Touyz RM, Yao G, Quinn MT, Pagano PJ, Schiffrin EL. p47phox
associates with the cytoskeleton through cortactin in human vascular smooth muscle cells: role in NAD(P)H oxidase regulation by angiotensin II. Arterioscler Thromb Vasc Biol. 2005 Mar ; 25(3):512-8.

Sawyer DB, Siwik DA, Xiao L, et al. Role of oxidative stress in
myocardial hypertrophy and failure. J Mol Cell Cardiol. 2002 ; 34: 379–388.

Takemoto M, Node K, Nakagami H, et al. Statins as antioxidant therapy
for preventing cardiac myocyte hypertrophy. J Clin Invest. 2001 ; 108: 1429–1437.

Luo JD, Xie F, Zhang WW, et al. Simvastatin inhibits
noradrenaline-induced hypertrophy of cultured neonatal rat cardiomyocytes. Br J Pharmacol. 2001 ; 132: 159–164.

Hirotani S, Otsu K, Nishida K, et al. Involvement of nuclear factor- B
and apoptosis signal-regulating kinase 1 in G-protein–coupled receptor agonist–induced cardiomyocyte hypertrophy. Circulation. 2002 ; 105: 509–515.

Shyu KG, Chen JJ, Shih NL, et al. Angiotensinogen gene expression is
induced by cyclical mechanical stretch in cultured rat cardiomyocytes. Biochem Biophys Res Commun. 1995 ; 211:241–248.

Shyu KG, Chang ML, Wang BW, et al. Cyclical mechanical stretching
increases the expression of vascular endothelial growth factor in rat vascular smooth cells.Formos Med Assoc. 2001 ; 100:741-747.

Bousette N, Giaid A. Urotensin-II and cardiovascular diseases. Curr
Hypertens Rep. 2006 Dec ; 8(6):479-83.

Zhu YC, Zhu YZ, Moore PK. The role of urotensin II in cardiovascular
and renal physiology and diseases. Br J Pharmacol.2006 Aug ; 148(7):884-901.

Rossowski WJ, Cheng BL, Taylor JE, Datta R, Coy DH. Human
urotensin II-induced aorta ring contractions are mediated by protein kinase C, tyrosine kinases and Rho-kinase: inhibition by somatostatin receptor antagonists. Eur J Pharmacol. 2002 Mar 8 ; 438(3):159-70

Watanabe T, Kanome T, Miyazaki A, Katagiri T. Human urotensin II as a
link between hypertension and coronary artery disease. Hypertens Res. 2006 Jun ; 29(6):375-87.

Watanabe T, Takahashi K, Kanome T, Hongo S, Miyazaki A, Koba S,
Katagiri T, Pakara R, Benedict CR. Human urotensin-II potentiates the mitogenic effect of mildly oxidized low-density lipoprotein on vascular smooth muscle cells: comparison with other vasoactive agents and hydrogen peroxide. Hypertens Res. 2006 Oct ; 29(10):821-31.

Chang H, Wang BW, Kuan P, Shyu KG. Cyclical mechanical stretch
enhances angiopoietin-2 and Tie2 receptor expression in cultured human umbilical vein endothelial cells. Clin Sci. 2003 ; 104:421-428.

Chang H, Shyu KG, Wang BW, Kuan P. Regulation of hypoxia-inducible
Factor-1alpha by cyclical mechanical stretch in rat vascular smooth muscle cells. Clin Sci (Lond). 2003 ; 105:447-456.

Cheng JJ, Wung BS, Chao YJ, Wang DL. Cyclical strain enhances
adhesion of monocytes to endothelial cells by increasing intercellular adhesion molecule-1 expression. Hypertension. 1996 ; 28:386-391.

Hu Y, Cheng L, Hochleitner BW, Xu Q. Activation of mitogen-activated
protein kinases (ERK/JNK) and AP-1 transcription factor in rat carotid arteries after balloon injury. Arterioscler Thromb Vasc Biol.1997 ; 17:2808-2816.



Li C, Hu Y, Mayr M, Xu Q. Cyclic strain stress-induced
mitogen-activated protein kinase (MAPK) phosphatase 1 expression in vascular smooth muscle cells is regulated by Ras/Rac-MAPK pathways. J Biol Chem. 1999 ; 274:25273-25280.

Locatelli V, Rossoni G, Schweiger F, Torsello A, De Gennaro Colonna
V,Bernareggi M, Deghenghi R, Muller EE, Berti F. Growth hormone-independent cardioprotective effects of hexarelin in the rat. Endocrinology. 1999 ; 140:4024-4031.

Shyu KG, Ko WH, Yang WS, Wang BW, Kuan P. Insulin-like growth
factor-1 mediates stretched-induced upregulation of myostatin expression in neonatal rat cardiomyocytes. Cardiovasc Res. 2005 ; 68:405-414.

Griendling KK, Sorescu D, Lassègue B, Ushio-Fukai M. Modulation of
protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology. Arterioscler Thromb Vasc Biol. 2000 Oct ; 20(10):2175-83.

Viedt C, Soto U, Krieger-Brauer HI, Fei J, Elsing C, Kübler W, Kreuzer.
Differential activation of mitogen-activated protein kinases in smooth muscle cells by angiotensin II: involvement of p22phox and reactive oxygen species. Arterioscler Thromb Vasc Biol. 2000 Apr ; 20(4):940-8.

Nakamura K, Fushimi K, Kouchi H, et al. Inhibitory effects of
antioxidants on neonatal rat cardiac myocyte hypertrophy induced by tumor necrosis factor- and angiotensin II. Circulation. 1998 ; 98: 794–799.

Wang DL, Tang CC, Wung BS, et al. Cyclical strain increases
endothelin-1 secretion and gene expression in human endothelial cells. Biochem Biophys Res Commun. 1995 ; 195:1050–1056.

Sudhir K, Wilson E, Chatterjee K, et al. Mechanical strain and collagen
potentiate activity of angiotensin II in rat vascular smooth muscle cells. J Clin Invest. 1993 ; 92:3003–3007.

Hung HF, Wang BW, Chang H, Shyu KG. The molecular regulation of
resistin expression in cultured vascular smooth muscle cells under hypoxia. J Hypertens. 2008 Dec ; 26(12):2349-60.
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