研究背景與目的：過去臨床追蹤研究發現，心肌在接受非標的放射照射後，會發生放射性心臟疾病。而在過去動物研究中，前置性有氧運動訓練對如缺血再灌流之心臟損傷具降低傷害之效益；但是否對接受放射照射之心臟也具相似的保護效益並不清楚。因此本研究的目的是以動物模式探討前置性有氧運動訓練是否能預防放射導致的心臟功能異常並探討可能之作用機制。
研究方法：本研究將24隻3至4週大之雄性Wistar大鼠，隨機分配到控制組（Ctrl）、放射組（IR）、運動訓練組（Ex）、及運動放射組（ExIR）（n=6）。Ex及ExIR組進行6週，每週5天，每天60分鐘之運動訓練。IR及ExIR大鼠在ExIR組完成運動訓練後，進行心臟分次放射照射，劑量為2 Gy/天，5天總量共10 Gy。所有大鼠在放射照射（或無照射）完成後一週，以導電心導管進行活體心臟功能測試（壓力-容積分析）。待分析結束後犧牲大鼠，取其左心室組織進行後續生化分析。以γH2AX分析脫氧核醣核酸；蛋白質之氧化傷害則以蛋白質羰基濃度分析；抗氧化能力以CuZnSOD及MnSOD mRNA表現量分析；以Masson''s trichrome染色觀察纖維化變化。
結果：相較於對照控制組，放射照射會造成心臟收縮及舒張功能顯著下降（兩者皆p<0.01），γH2AX發生率（Ctrl: 23.3% vs. IR: 38.9%, p<0.01）及蛋白質羰基濃度（Ctrl: 2.3 nmol/mg vs. IR: 3.5 nmol/mg, p<0.01）顯著較高。相較於無運動訓練之對照組，前置性運動訓練後進行放射照射之大鼠，心臟收縮及舒張功能下降之情形顯著較小（兩者皆p<0.01），γH2AX發生率（IR: 38.9% vs. ExIR: 27.3%, p<0.01）及蛋白質羰基濃度（IR: 3.5 nmol/mg vs. ExIR: 2.2 nmol/mg, p<0.01）顯著較低。相較於未訓練的對照組，運動訓練組的CuZnSOD mRNA表現量（IR: 0.3 vs. ExIR: 0.5, p<0.01）及MnSOD mRNA表現量（IR: 0.1 vs. ExIR: 0.9, p<0.01）皆顯著較高。纖維化變化分析在各組之間未見顯著差異。
結論：放射照射後一週會造成心臟收縮及舒張功能異常，而前置性有氧運動訓練可透過正調控心肌之抗氧化能力來達到心臟保護效益。

Background and purpose: Clinical studies have reported that cardiac irradiation during treatment of thoracic cancers could lead to radiation-induced heart disease. Exercise preconditioning has been shown to decrease ischemia-reperfusion related cardiac damage in animal studies. Whether exercise preconditioning could ameliorate irradiation induced cardiac injury remains to be determined. The aims of this study were to investigate the effects of preconditioned exercise training on irradiation related cardiac dysfunction and exploring the potential underlying mechanism in animal model.
Methods: A total of 24 adult male Wistar rats aged 3 to 4 week-old were randomized into control (Ctrl), irradiation (IR), exercise training (Ex) and exercise training + irradiation (ExIR) group (n=6 per group). The Ex and ExIR underwent exercise training on a motor-driven treadmill for 60 minutes/day, 5 days/week, for a total of 6 weeks. After anesthetized, rats in IR and ExIR received 2 Gy/day irradiation to the heart region for 5 days (total dose of 10 Gy). In vivo cardiac function (pressure-volume analysis) were examined one week after all designated interventions were complete and left ventricles were excised for biochemical analysis. The mRNA levels of antioxidant enzyme (i.e., CuZnSOD and MnSOD) were determined using quantitative real-time PCR analysis. Retention of γH2AX foci was analyzed and used as an indicator for DNA damage. Level of protein carbonyl was analyzed as an indicator for oxidative injury to protein. Masson’s trichrome staining was used to exam myocardial fibrosis.
Results: Compared to no irradiation Ctrl, cardiac systolic and diastolic function were significantly lower (both p<0.01), γH2AX (Ctrl: 23.3% vs. IR: 38.9%, p<0.01) and protein carbonyl (Ctrl: 2.3 nmol/mg vs. IR: 3.5 nmol/mg, p<0.01) was significantly higher in IR group. Compared to no exercise training, rats in ExIR group had better cardiac systolic and diastolic function (both p<0.01) after irradiation, and had significantly lower γH2AX (IR: 38.9% vs. ExIR: 27.3%, p<0.01) and protein carbonyl (IR: 3.5 nmol/mg vs. ExIR: 2.2 nmol/mg, p<0.01). CuZnSOD mRNA (IR: 0.3 vs. ExIR: 0.5, p<0.01) and MnSOD mRNA (IR: 0.1 vs. ExIR: 0.9, p<0.01) were significantly higher in the ExIR group compared to those of IR group. No apparent fibrotic changes was observed in all experiment groups.
Conclusion: The results of this study showed that exercise preconditioning could ameliorate cardiac dysfunction after irradiation through upregulation its antioxidant capacity.

致謝 I
中文摘要 II
Abstract IV
目錄 VI
第一章、前言 1
第一節、研究背景與目的 1
第二節、研究假說 1
第三節、研究重要性 2
第二章、文獻回顧 3
第一節、放射治療 3
第二節、放射治療之心臟毒性 4
第三節、放射治療引起心臟毒性之機制 7
第四節、運動誘發之心臟保護作用 8
第三章、研究方法及實驗步驟 11
第一節、研究設計 11
第二節、研究動物 11
第三節、樣本數估計 11
第四節、實驗流程 11
第五節、有氧運動訓練 12
第六節、放射照射方法及其參數設定 13
第七節、導電心導管壓力體積曲線測量 13
第八節、研究工具及方法 14
第九節、研究變項 22
第十節、統計分析 23
第四章、結果 25
第一節、基本資料 25
第二節、放射照射及運動訓練對心臟功能之影響 25
第三節、放射照射對心臟組織之氧化傷害影響 26
第四節、運動訓練對心臟組織之抗氧化能力影響 27
第五節、放射照射對心臟產生之發炎現象變化 27
第六節、放射照射對心臟組織之纖維化影響 27
第五章、討論 28
第六章、結論 33
參考資料 34
圖1實驗流程圖 40
圖2實驗動物擺位及放射照射示意圖 41
圖3 DNA傷害γH2AX染色示意圖 42
圖4蛋白質羰基濃度含量分析 43
圖5 CuZnSOD mRNA含量分析 44
圖6 MnSOD mRNA含量分析 45
圖7發炎現象H＆E染色示意圖 46
圖8纖維化Masson’s trichrome染色示意圖 47
表1有氧運動訓練計畫表 48
表2導電心導管壓力體積曲線測量一般參數結果 49
表3導電心導管壓力體積曲線測量收縮參數結果 50
表4導電心導管壓力體積曲線測量舒張參數結果 51

1.衛生福利部統計處. 民國107年主要死因分析. 2018.
2.衛生福利部國民健康署. 癌症登記報告. 2018.
3.Cox JD, Ang KK. Radiation Oncology: Rationale, Technique, Results: Elsevier Health Sciences; 2009;10-12.
4.Ikushima H. Radiation therapy: state of the art and the future. J Med Investig. 2010;57:1-11.
5.Jaworski C, Mariani JA, Wheeler G, Kaye DM. Cardiac complications of thoracic irradiation. J Am Coll Cardiol. 2013;61:2319-28.
6.Lund M, von Döbeln GA, Nilsson M, Winter R, Lundell L, Tsai JA, et al. Effects on heart function of neoadjuvant chemotherapy and chemoradiotherapy in patients with cancer in the esophagus or gastroesophageal junction–a prospective cohort pilot study within a randomized clinical trial. Radiat Oncol. 2015;10:16.
7.Chargari C, Kirov KM, Bollet MA, Magné N, Védrine L, Cremades S, et al. Cardiac toxicity in breast cancer patients: from a fractional point of view to a global assessment. Cancer Treat Rev. 2011;37:321-30.
8.Henning RJ, Harbison RD. Cardio-oncology: cardiovascular complications of cancer therapy. Future Cardiol. 2017;13:379-96.
9.Zhao W, Robbins ME. Inflammation and chronic oxidative stress in radiation-induced late normal tissue injury: therapeutic implications. Curr Med Chem. 2009;16:130-43.
10.French JP, Hamilton KL, Quindry JC, Lee Y, Upchurch PA, Powers SK. Exercise-induced protection against myocardial apoptosis and necrosis: MnSOD, calcium-handling proteins, and calpain. FASEB J. 2008;22:2862-71.
11.Ewer MS, Ewer SM. Cardiotoxicity of anticancer treatments. Nat Rev Cardiol. 2015;12:547.
12.Lu W, Chen M, Chen Q, Ruchala K, Olivera G. Adaptive fractionation therapy: I. Basic concept and strategy. Phys Med Biol. 2008;53:5495.
13.Groarke JD, Nguyen PL, Nohria A, Ferrari R, Cheng S, Moslehi J. Cardiovascular complications of radiation therapy for thoracic malignancies: the role for non-invasive imaging for detection of cardiovascular disease. Eur Heart J. 2013;35:612-23.
14.Clark R, Berry N, Chowdhury M, McCarthy A, Ullah S, Versace V, et al. Heart failure following cancer treatment: characteristics, survival and mortality of a linked health data analysis. Intern Med J. 2016;46:1297-306.
15.Powers SK, Quindry JC, Kavazis AN. Exercise-induced cardioprotection against myocardial ischemia–reperfusion injury. Free Radic Biol Med. 2008;44:193-201.
16.Darby SC, Cutter DJ, Boerma M, Constine LS, Fajardo LF, Kodama K, et al. Radiation-related heart disease: current knowledge and future prospects. Int J Radiat Oncol Biol Phys. 2010;76:656-65.
17.Galper SL, James BY, Mauch PM, Strasser JF, Silver B, LaCasce A, et al. Clinically significant cardiac disease in patients with Hodgkin lymphoma treated with mediastinal irradiation. Blood. 2011;117:412-8.
18.Gottdiener JS, Katin MJ, Borer JS, Bacharach SL, Green MV. Late cardiac effects of therapeutic mediastinal irradiation: assessment by echocardiography and radionuclide angiography. N Engl J Med. 1983;308:569-72.
19.Schubert LK, Gondi V, Sengbusch E, Westerly DC, Soisson ET, Paliwal BR, et al. Dosimetric comparison of left-sided whole breast irradiation with 3DCRT, forward-planned IMRT, inverse-planned IMRT, helical tomotherapy, and topotherapy. Radiother Oncol. 2011;100:241-6.
20.Nellessen U, Zingel M, Hecker H, Bahnsen J, Borschke D. Effects of radiation therapy on myocardial cell integrity and pump function: which role for cardiac biomarkers? Chemotherapy. 2010;56:147-52.
21.Jagsi R, Moran J, Marsh R, Masi K, Griffith KA, Pierce LJ. Evaluation of four techniques using intensity-modulated radiation therapy for comprehensive locoregional irradiation of breast cancer. Int J Radiat Oncol Biol Phys. 2010;78:1594-603.
22.Aznar MC, Korreman S, Pedersen AN, Persson GF, Josipovic M, Specht L. Evaluation of dose to cardiac structures during breast irradiation. Br J Radiol. 2011;84:743-6.
23.Preston DL, Shimizu Y, Pierce DA, Suyama A, Mabuchi K. Studies of mortality of atomic bomb survivors. Report 13: solid cancer and noncancer disease mortality: 1950–1997. Radiat Res. 2012;178:AV146-AV72.
24.Mezzaroma E, Di X, Graves P, Toldo S, Van Tassell BW, Kannan H, et al. A mouse model of radiation-induced cardiomyopathy. Int J Cardiol. 2012;156:231-3.
25.Boerma M, Roberto KA, Hauer-Jensen M. Prevention and treatment of functional and structural radiation injury in the rat heart by pentoxifylline and alpha-tocopherol. Int J Radiat Oncol Biol Phys. 2008;72:170-7.
26.Hendry JH, Akahoshi M, Wang LS, Lipshultz SE, Stewart FA, Trott KR. Radiation-induced cardiovascular injury. Radiat Environ Biophys. 2008;47:189-93.
27.Boerma M, Zurcher C, Esveldt I, Schutte-Bart C, Wondergem J. Histopathology of ventricles, coronary arteries and mast cell accumulation in transverse and longitudinal sections of the rat heart after irradiation. Oncol Rep. 2004;12:213-9.
28.Magné N, Melis A, Chargari C, Castadot P, Guichard J-B, Barani D, et al. Recommendations for a lifestyle which could prevent breast cancer and its relapse: Physical activity and dietetic aspects. Crit Rev Oncol Hematol. 2011;80:450-9.
29.Ewer M, Gianni L, Pane F, Sandri MT, Steiner RK, Wojnowski L, et al. Report on the international colloquium on cardio-oncology (rome, 12–14 march 2014). Ecancermedicalscience. 2014;8:433.
30.Madan R, Benson R, Sharma D, Julka P, Rath G. Radiation induced heart disease: pathogenesis, management and review literature. J Natl Cancer I. 2015;27:187-93.
31.Wu Q-Q, Xiao Y, Yuan Y, Ma Z-G, Liao H-H, Liu C, et al. Mechanisms contributing to cardiac remodelling. Clin Sci. 2017;131:2319-45.
32.Murtha LA, Schuliga MJ, Mabotuwana NS, Hardy SA, Waters DW, Burgess JK, et al. The processes and mechanisms of cardiac and pulmonary fibrosis. Front Physiol. 2017;8:777.
33.Hudson SV, Dolin CE, Poole LG, Massey VL, Wilkey D, Beier JI, et al. Modeling the kinetics of integrin receptor binding to hepatic extracellular matrix proteins. Sci Rep. 2017;7:12444.
34.Gürses İ, Özeren M, Serin M, Yücel N, Erkal HŞ. Histopathological evaluation of melatonin as a protective agent in heart injury induced by radiation in a rat model. Pathol Res Pract. 2014;210:863-71.
35.Filopei J, Frishman W. Radiation-induced heart disease. Cardiol Rev. 2012;20:184-8.
36.Laughlin MH, Bowles DK, Duncker DJ. The coronary circulation in exercise training. Am J Physiol Heart Circ Physiol. 2011;302:H10-H23.
37.Senf SM, Dodd SL, McClung JM, Judge AR. Hsp70 overexpression inhibits NF-κB and Foxo3a transcriptional activities and prevents skeletal muscle atrophy. FASEB J. 2008;22:3836-45.
38.Naito H, Powers SK, Demirel HA, Aoki J. Exercise training increases heat shock protein in skeletal muscles of old rats. Med Sci Sports Exerc. 2001;33:729-34.
39.Quindry JC, Hamilton KL, French JP, Lee Y, Murlasits Z, Tumer N, et al. Exercise-induced HSP-72 elevation and cardioprotection against infarct and apoptosis. J Appl Physiol. 2007;103:1056-62.
40.Kavazis AN. Exercise preconditioning of the myocardium. Sports Med. 2009;39:923-35.
41.Calvert JW, Lefer DJ. Role of β-adrenergic receptors and nitric oxide signaling in exercise-mediated cardioprotection. Physiology. 2013;28:216-24.
42.Powers SK, Jackson MJ. Exercise-induced oxidative stress: cellular mechanisms and impact on muscle force production. Physiol Rev. 2008;88:1243-76.
43.Ansley DM, Wang B. Oxidative stress and myocardial injury in the diabetic heart. J Pathol. 2013;229:232-41.
44.Powers SK, Smuder AJ, Kavazis AN, Quindry JC. Mechanisms of exercise-induced cardioprotection. Physiology. 2014;29:27-38.
45.Kavazis AN, Alvarez S, Talbert E, Lee Y, Powers SK. Exercise training induces a cardioprotective phenotype and alterations in cardiac subsarcolemmal and intermyofibrillar mitochondrial proteins. Am J Physiol Heart Circ Physiol. 2009;297:H144-H52.
46.Ma X, Fu Y, Xiao H, Song Y, Chen R, Shen J, et al. Cardiac fibrosis alleviated by exercise training is AMPK-dependent. PloS one. 2015;10:e0129971.
47.Pacher P, Nagayama T, Mukhopadhyay P, Bátkai S, Kass DA. Measurement of cardiac function using pressure–volume conductance catheter technique in mice and rats. Nat Protoc. 2008;3:1422.
48.Tapio S. Pathology and biology of radiation-induced cardiac disease. J Radiat Res. 2016;57:439-48.
49.Heidenreich PA, Hancock SL, Vagelos RH, Lee BK, Schnittger I. Diastolic dysfunction after mediastinal irradiation. Am Heart J. 2005;150:977-82.
50.Hu S, Chen Y, Li L, Chen J, Wu B, Zhou X, et al. Effects of adenovirus-mediated delivery of the human hepatocyte growth factor gene in experimental radiation-induced heart disease. Int J Radiat Oncol Biol Phys. 2009;75:1537-44.
51.Boerma M, Wang J, Kulkarni A, Roberto KA, Qiu X, Kennedy RH, et al. Influence of endothelin 1 receptor inhibition on functional, structural and molecular changes in the rat heart after irradiation. Radiat Res. 2008;170:275-83.
52.Cilliers G, Harper I, Lochner A. Radiation-induced changes in the ultrastructure and mechanical function of the rat heart. Radiother Oncol 1989;16:311-26.
53.Rübe CE, Grudzenski S, Kühne M, Dong X, Rief N, Löbrich M, et al. DNA double-strand break repair of blood lymphocytes and normal tissues analysed in a preclinical mouse model: implications for radiosensitivity testing. Clin Cancer Res. 2008;14:6546-55.
54.Gavrilov B, Vezhenkova I, Firsanov D, Solovjeva L, Svetlova M, Mikhailov V, et al. Slow elimination of phosphorylated histone γ-H2AX from DNA of terminally differentiated mouse heart cells in situ. Biochem Biophys Res Commun. 2006;347:1048-52.
55.Park SH, Pradeep K, Ko KC. Protective effect of hesperidin against γ-radiation induced oxidative stress in Sprague-Dawley rats. Pharm Biol. 2009;47:940-7.
56.Havaki S, Kotsinas A, Chronopoulos E, Kletsas D, Georgakilas A, Gorgoulis VG. The role of oxidative DNA damage in radiation induced bystander effect. Cancer Lett. 2015;356:43-51.
57.Azimzadeh O, Scherthan H, Sarioglu H, Barjaktarovic Z, Conrad M, Vogt A, et al. Rapid proteomic remodeling of cardiac tissue caused by total body ionizing radiation. Proteomics. 2011;11:3299-311.
58.Kruse JJCM, Floot BG, te Poele JA, Russell NS, Stewart FA. Radiation-induced activation of TGF-β signaling pathways in relation to vascular damage in mouse kidneys. Radiat Res. 2009;171:188-97.
59.Gujral DM, Lloyd G, Bhattacharyya S. Radiation-induced valvular heart disease. Heart. 2016;102:269-76.
60.Wang J, Wu Y, Yuan F, Liu Y, Wang X, Cao F, et al. Chronic intermittent hypobaric hypoxia attenuates radiation induced heart damage in rats. Life Sci. 2016;160:57-63.
61.Saiki H, Moulay G, Guenzel AJ, Liu W, Decklever TD, Classic KL, et al. Experimental cardiac radiation exposure induces ventricular diastolic dysfunction with preserved ejection fraction. Am J Physiol Heart Circ Physiol. 2017;313:H392-H407.
62.Baker JE, Fish BL, Su J, Haworth ST, Strande JL, Komorowski RA, et al. 10 Gy total body irradiation increases risk of coronary sclerosis, degeneration of heart structure and function in a rat model. Int J Radiat Biol. 2009;85:1089-100.
63.Hydock DS, Lien C-Y, Jensen BT, Schneider CM, Hayward R. Exercise preconditioning provides long-term protection against early chronic doxorubicin cardiotoxicity. Integr Cancer Ther. 2011;10:47-57.
64.Thorp DB, Haist JV, Leppard J, Milne KJ, Karmazyn M, Noble EG. Exercise training improves myocardial tolerance to ischemia in male but not in female rats. Am J Physiol Regul Integr Comp Physiol. 2007;293:R363-R71.
65.Powers SK, Criswell D, Lawler J, Martin D, Lieu F, Ji LL, et al. Rigorous exercise training increases superoxide dismutase activity in ventricular myocardium. Am J Physiol Heart Circ Physiol. 1993;265:H2094-H8.
66.Bowles D, Starnes JW. Exercise training improves metabolic response after ischemia in isolated working rat heart. J Appl Physiol. 1994;76:1608-14.
67.Bowles D, Farrar R, Starnes JW. Exercise training improves cardiac function after ischemia in the isolated, working rat heart. Am J Physiol Heart Circ Physiol. 1992;263:H804-H9.
68.Dolinsky VW, Rogan KJ, Sung MM, Zordoky BN, Haykowsky MJ, Young ME, et al. Both aerobic exercise and resveratrol supplementation attenuate doxorubicin-induced cardiac injury in mice. Am J Physiol Endocrinol Metab. 2013;305:E243-E53.
69.Kavazis AN, Smuder AJ, Min K, Tümer N, Powers SK. Short-term exercise training protects against doxorubicin-induced cardiac mitochondrial damage independent of HSP72. Am J Physiol Heart Circ Physiol. 2010;299:H1515-H24.
70.Fukai T, Ushio-Fukai M. Superoxide dismutases: role in redox signaling, vascular function, and diseases. Antioxid Redox Signal. 2011;15:1583-606.
71.Li Y, Huang T-T, Carlson EJ, Melov S, Ursell PC, Olson JL, et al. Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase. Nat Genet. 1995;11:376.
72.Chen Z, Siu B, Ho Y-S, Vincent R, Chua CC, Hamdy RC, et al. Overexpression of MnSOD protects against myocardial ischemia/reperfusion injury in transgenic mice. J Mol Cell Cardiol. 1998;30:2281-9.
73.Yamashita N, Hoshida S, Otsu K, Asahi M, Kuzuya T, Hori M. Exercise provides direct biphasic cardioprotection via manganese superoxide dismutase activation. J Exp Med. 1999;189:1699-706.
74.Lee Y, Min K, Talbert EE, Kavazis AN, Smuder AJ, Willis WT, et al. Exercise protects cardiac mitochondria against ischemia-reperfusion injury. Med Sci Sports Exerc. 2012;44:397-405.