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研究生:許智超
研究生(外文):Chih-Chao Hsu
論文名稱:運動訓練對卵巢切除之大鼠心臟凋亡以及抗凋亡的影響
論文名稱(外文):Effect of exercise training on cardiac apoptosis and survival pathways in ovariectomized rats
指導教授:李信達李信達引用關係
指導教授(外文):Shin-Da Lee
學位類別:碩士
校院名稱:中國醫藥大學
系所名稱:物理治療學系復健科學碩士班
學門:醫藥衛生學門
學類:復健醫學學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:65
中文關鍵詞:心臟卵巢切除細胞凋亡停經
外文關鍵詞:heartTNF-αcaspasescell deathOvariectomy
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研究資料顯示停經或卵巢切除後的婦女大幅提高了心臟疾病的風險。過去研究發現在卵巢切除的大鼠身上有心肌細胞凋亡的現象,然而對於停經或是卵巢切除後的女性運動對心肌細胞凋亡的影響方面的研究並不多。本篇的目的便在研究雙側卵巢切除後的大鼠經由運動訓練後對其Fas蛋白和粒線體依賴性心臟細胞凋亡以及存活和抗凋亡途徑的影響。實驗使用三十三隻大鼠,其中十一隻手術過程中不切除卵巢作為對照組,另外二十二隻卵巢切除大鼠中再隨機挑選十一隻於休息一周後開始進行十周的運動訓練。在訓練完成後將三十三隻大鼠犧牲取出心臟並測量heart index、hematoxylin-eosin staining、Western Blotting 以及 positive TUNEL assays。實驗結果卵巢切除組大鼠在心臟重、不正常的心肌組織、Fas以及粒線體依賴性心臟細胞凋亡途徑表現蛋白以及TUNEL陽性細胞,相較於對照組都有明顯的增加;而這些在運動組相較於雙側卵巢切除組則有明顯的下降。藉由本實驗證明了運動可以抑制或是預防卵巢切除所導致的Fas蛋白以及粒線體依賴性心臟細胞凋亡,並增進存活途徑的保護作用。由於雌激素與其替代品的補充治療有提高乳癌發生的風險,因此這項發現在停經或是卵巢切除後的婦女藉由運動預防心血管疾病上提供了一個新的治療效果。

Background. Cardiac apoptosis were found in ovariectomized rats but very limited information regarding the effects of exercise training on cardiac apoptosis in menopausal or bilateral oophorectomied women was available. The purpose of this study was to evaluate the effects of exercise training on cardiac Fas and mitochondria dependent apoptotic pathways and cardiac survival pathways in ovariectomized rats. Methods. Eleven sham-operated rats(Sham) and eleven ovariectomized rats(OVX) at 14 weeks of age were served as negative and positive control. Eleven ovariectomized rats underwent treadmill running exercise 1 hour daily for 10 weeks(OVX-EX). After exercise training or sedentary status, the excised hearts were measured by heart index, hematoxylin-eosin staining, Western Blotting and positive TUNEL assays. Results. The whole heart weight, the left ventricular weight, the ratios of whole heart weight to tibia length, and the ratios of left ventricle to tibia length were significantly increased in OVX relative to Sham. Abnormal myocardial architecture and more cardiac TUNEL-positive apoptotic cells were observed in OVX, but not in Sham. Cardiac Fas ligand, Fas death receptors, Fas-associated death domain (FADD), t-Bid, Bad, Bak, Bax, activated caspase 8, activated caspase 9, and activated caspase 3 in OVX were significantly increased, compared to Sham. OVX-induced protein levels of TNF-alpha, Fas ligand, Fas death receptors, FADD, activated caspase 8, and activated caspase 3 (Fas pathways) became lower in OVX-EX. OVX-induced protein levels of t-Bid, Bad, Bax, Bak, activated caspase 9, and activated caspase 3 (mitochondria pathway) became lower in OVX-EX. Furthermore Cardiac phosphorylated PI3K, phosphorylated AKT, phosphorylated p38, Bcl2 and phosphorylated Bad were significanly increased in OVX-EX, compared to OVX. Conclusions. Exercise training suppressed ovariectomy-induced cardiac Fas and mitochondria dependent apoptotic pathways, and furthermore it increase cardiac survival pathways in ovariectomized rat models. The findings may provide one of new therapeutic effect of exercise training on preventing cardiac apoptosis in
iii
menopausal or bilateral oophorectomied women.

Part 1.
Effect of exercise training on cardiac Fas and mitochondria dependant apoptosis pathways in ovariectomized rats …………………………………….1
Introduction ………………………………………………………………….…….……….2
Materials and Methods ………………………………………………………….…….5
Animal model
Exercise training
Blood Pressure and Echocardiography
Cardiac characteristics
Tissue Extraction
Electrophoresis and Western Blot
Hematoxylin-eosin staining (H&E staining) and Terminal Deoxynucleotide
Transferase-mediated dUTP Nick End Labeling (TUNEL)
Statistical Analysis
Results …………………………………………………………………....……………….10
Discussion ……………………………………………………………..….……………..13
References ………………………………………………………….…..…….…………17
Table …………….…………………………………………………….…..…….…………21
Figures ……………………….………………………………………….………....…….22
v
TABLE AND FIGURE CONTENTS
Table 1. Heart weight index ……….21
Fig 1. Hematoxylin and eosin staining was analyzed in cardiac sections ………..22
Fig 2. The representative protein products of Fas and Fas ligand ………..23
Fig 3. The representative protein products of TNFα and TNFR1 ………..24
Fig 4. The representative protein products of FADD ………..25
Fig 5. The representative protein products of t-Bid ...……..26
Fig 6. The representative protein products of Bad ………..27
Fig 7. The representative protein products of Bak and Bax ………..28
Fig 8. The representative protein products of cytochrome c ………..29
Fig 9. The representative protein products of caspase 8 ………..30
Fig 10. The representative protein products of caspase 9 ………..31
Fig 11. The representative protein products of caspase 3 ………..32
Fig 12. Cardiac TUNEL assay ………..33
Fig 13. Cardiac Fas and mitochondria dependent apoptotic pathways ………..34
vi
Part 2.
Effect of exercise training on cardiac survival pathways in ovariectomized rats ………………………………………………………………………………………….36
Introduction ………………………………………………………………….…….……….37
Materials and Methods …………………………………………………….…….……40
Animal model
Exercise training
Blood Pressure and Echocardiography
Cardiac characteristics
Tissue Extraction
Electrophoresis and Western Blot
Hematoxylin-eosin staining (H&E staining) and Terminal Deoxynucleotide
Transferase-mediated dUTP Nick End Labeling (TUNEL)
Statistical Analysis
Results …………………………………………………………………....………..……45
Discussion ……………………………………………………………………….………47
References ………………………………………………………….………….…….…50
Table ………………………………………………………….………….……………..…55
Figures ………………………………………………………….………………..………56
vii
TABLE AND FIGURE CONTENTS
Table 1. Heart weight index ……….55
Fig 1. Hematoxylin and eosin staining was analyzed in cardiac sections ………..56
Fig 2. The representative protein products of IGF1 and IGF1R ………..57
Fig 3. The representative protein products of p-PI3K and PI3K ………..58
Fig 4. The representative protein products of p-AKT and AKT ...……..59
Fig 5. The representative protein products of p-P38 and P38 ………..60
Fig 6. The representative protein products of Bcl-2 ………..61
Fig 7. The representative protein products of p-Bad ………..62
Fig 8. Cardiac TUNEL assay ………..63
Fig 9. Cardiac survival pathways ………..64

Part 1.
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18
[12]L.P. Grazette, A. Rosenzweig, Role of apoptosis in heart failure. Heart Fail Clin 1 (2005) 251-261.
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19
Liu, M.H. Chang, C.Y. Huang, Effects of long-term intermittent hypoxia on mitochondrial and Fas death receptor dependent apoptotic pathways in rat hearts. Int J Cardiol 116 (2007) 348-356.
[26]S.D. Lee, W.W. Kuo, C.H. Wu, Y.M. Lin, J.A. Lin, M.C. Lu, A.L. Yang, J.Y. Liu, S.G. Wang, C.J. Liu, L.M. Chen, C.Y. Huang, Effects of short- and long-term hypobaric hypoxia on Bcl2 family in rat heart. Int J Cardiol 108 (2006) 376-384.
[27]S.D. Lee, B.S. Tzang, W.W. Kuo, Y.M. Lin, A.L. Yang, S.H. Chen, F.J. Tsai, F.L. Wu, M.J. Lu, C.Y. Huang, Cardiac fas receptor-dependent apoptotic pathway in obese Zucker rats. Obesity (Silver Spring) 15 (2007) 2407-2415.
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Part 2.
[1]L. Mosca, S.M. Grundy, D. Judelson, K. King, M. Limacher, S. Oparil, R. Pasternak, T.A. Pearson, R.F. Redberg, S.C. Smith, Jr., M. Winston, S. Zinberg, AHA/ACC scientific statement: consensus panel statement. Guide to preventive cardiology for women. American Heart Association/American College of Cardiology. J Am Coll Cardiol 33 (1999) 1751-1755.
[2]A. Zanesco, P.R. Zaros, [Physical exercise and menopause]. Rev Bras Ginecol Obstet 31 (2009) 254-261.
[3]D.C. Skegg, Hormone therapy and heart disease after the menopause. Lancet 358 (2001) 1196-1197.
[4]E. Lokkegaard, Z. Jovanovic, B.L. Heitmann, N. Keiding, B. Ottesen, A.T. Pedersen, The association between early menopause and risk of ischaemic heart disease: influence of Hormone Therapy. Maturitas 53 (2006) 226-233.
[5]F. Atsma, M.L. Bartelink, D.E. Grobbee, Y.T. van der Schouw, Postmenopausal status and early menopause as independent risk factors for cardiovascular disease: a meta-analysis. Menopause 13 (2006) 265-279.
[6]M. Manco, G. Nolfe, M. Calvani, A. Natali, J. Nolan, E. Ferrannini, G. Mingrone, Menopause, insulin resistance, and risk factors for cardiovascular disease. Menopause 13 (2006) 809-817.
[7]R. Rossi, T. Grimaldi, G. Origliani, G. Fantini, F. Coppi, M.G. Modena, Menopause and cardiovascular risk. Pathophysiol Haemost Thromb 32 (2002) 325-328.
[8]M. Stramba-Badiale, K.M. Fox, S.G. Priori, P. Collins, C. Daly, I. Graham, B. Jonsson, K. Schenck-Gustafsson, M. Tendera, Cardiovascular diseases in women: a statement from the policy conference of the European Society of Cardiology. Eur Heart J 27 (2006) 994-1005.
[9]P.M. Rautaharju, C. Kooperberg, J.C. Larson, A. LaCroix, Electrocardiographic predictors of incident congestive heart failure and all-cause mortality in postmenopausal women: the Women''s Health Initiative. Circulation 113 (2006) 481-489.
[10]J. Narula, N. Haider, E. Arbustini, Y. Chandrashekhar, Mechanisms of disease: apoptosis in heart failure--seeing hope in death. Nat Clin Pract Cardiovasc Med 3 (2006) 681-688.
[11]S.D. Lee, W.W. Kuo, Y.J. Ho, A.C. Lin, C.H. Tsai, H.F. Wang, C.H. Kuo, A.L. Yang, C.Y. Huang, J.M. Hwang, Cardiac Fas-dependent and mitochondria-dependent apoptosis in ovariectomized rats. Maturitas 61 (2008) 268-277.
51
[12]L.P. Grazette, A. Rosenzweig, Role of apoptosis in heart failure. Heart Fail Clin 1 (2005) 251-261.
[13]M. Das, Apoptosis as a therapeutic target in heart failure. Am J Physiol Heart Circ Physiol 293 (2007) H1322-1323.
[14]A. Haunstetter, S. Izumo, Apoptosis: basic mechanisms and implications for cardiovascular disease. Circ Res 82 (1998) 1111-1129.
[15]S.D. Lee, C.H. Chu, E.J. Huang, M.C. Lu, J.Y. Liu, C.J. Liu, H.H. Hsu, J.A. Lin, W.W. Kuo, C.Y. Huang, Roles of insulin-like growth factor II in cardiomyoblast apoptosis and in hypertensive rat heart with abdominal aorta ligation. Am J Physiol Endocrinol Metab 291 (2006) E306-314.
[16]J. Narula, P. Pandey, E. Arbustini, N. Haider, N. Narula, F.D. Kolodgie, B. Dal Bello, M.J. Semigran, A. Bielsa-Masdeu, G.W. Dec, S. Israels, M. Ballester, R. Virmani, S. Saxena, S. Kharbanda, Apoptosis in heart failure: release of cytochrome c from mitochondria and activation of caspase-3 in human cardiomyopathy. Proc Natl Acad Sci USA 96 (1999) 8144-8149.
[17]N.H. Bishopric, P. Andreka, T. Slepak, K.A. Webster, Molecular mechanisms of apoptosis in the cardiac myocyte. Curr Opin Pharmacol 1 (2001) 141-150.
[18]D. Siegmund, D. Mauri, N. Peters, P. Juo, M. Thome, M. Reichwein, J. Blenis, P. Scheurich, J. Tschopp, H. Wajant, Fas-associated death domain protein (FADD) and caspase-8 mediate up-regulation of c-Fos by Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via a FLICE inhibitory protein (FLIP)-regulated pathway. J Biol Chem 276 (2001) 32585-32590.
[19]B.B. Aggarwal, U. Bhardwaj, Y. Takada, Regulation of TRAIL-induced apoptosis by ectopic expression of antiapoptotic factors. Vitam Horm 67 (2004) 453-483.
[20]B.C. Barnhart, E.C. Alappat, M.E. Peter, The CD95 type I/type II model. Semin Immunol 15 (2003) 185-193.
[21]Y. Tsujimoto, Role of Bcl-2 family proteins in apoptosis: apoptosomes or mitochondria? Genes Cells 3 (1998) 697-707.
[22]A. Gross, J.M. McDonnell, S.J. Korsmeyer, BCL-2 family members and the mitochondria in apoptosis. Genes Dev 13 (1999) 1899-1911.
[23]L.A. Kubasiak, O.M. Hernandez, N.H. Bishopric, K.A. Webster, Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNIP3. Proc Natl Acad Sci U S A 99 (2002) 12825-12830.
[24]B. Antonsson, Mitochondria and the Bcl-2 family proteins in apoptosis signaling pathways. Mol Cell Biochem 256-257 (2004) 141-155.
[25]S.D. Lee, W.W. Kuo, J.A. Lin, Y.F. Chu, C.K. Wang, Y.L. Yeh, S.G. Wang, J.Y.
52
Liu, M.H. Chang, C.Y. Huang, Effects of long-term intermittent hypoxia on mitochondrial and Fas death receptor dependent apoptotic pathways in rat hearts. Int J Cardiol 116 (2007) 348-356.
[26]S.D. Lee, W.W. Kuo, C.H. Wu, Y.M. Lin, J.A. Lin, M.C. Lu, A.L. Yang, J.Y. Liu, S.G. Wang, C.J. Liu, L.M. Chen, C.Y. Huang, Effects of short- and long-term hypobaric hypoxia on Bcl2 family in rat heart. Int J Cardiol 108 (2006) 376-384.
[27]S.D. Lee, B.S. Tzang, W.W. Kuo, Y.M. Lin, A.L. Yang, S.H. Chen, F.J. Tsai, F.L. Wu, M.J. Lu, C.Y. Huang, Cardiac fas receptor-dependent apoptotic pathway in obese Zucker rats. Obesity (Silver Spring) 15 (2007) 2407-2415.
[28]M.C. Lu, B.S. Tzang, W.W. Kuo, F.L. Wu, Y.S. Chen, C.H. Tsai, C.Y. Huang, S.D. Lee, More activated cardiac mitochondrial-dependent apoptotic pathway in obese Zucker rats. Obesity (Silver Spring) 15 (2007) 2634-2642.
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