臺灣博碩士論文加值系統

　　動脈粥狀硬化是工業化社會中的主要致死原因之一。運動已廣泛的被認為具有治療動脈粥狀硬化的功用。在其他的研究指出，長期的運動訓練可以降低骨骼肌細胞凋亡的情形和降低促細胞凋亡蛋白的表現。前人的研究證明,在動脈粥狀硬化的損傷區上有細胞凋亡和促細胞凋亡相關蛋白表現的情形，而細胞的凋亡會導致動脈粥狀硬化的惡化。然而，有關長期的運動訓練，對於動脈粥狀硬化血管之細胞凋亡相關蛋白表現，仍是不清楚的。因此本篇論文的目的，是要探討長期的運動訓練，對於血管細胞上的細胞凋亡相關蛋白表現的影響。採用ApoE基因剔除小鼠餵食0.15% 高膽固醇飼料當作動脈粥狀硬化的動物模式，給予ApoE基因剔除公鼠跑步機的運動訓練，每天兩次，一次60分鐘 (速度為每分鐘15公尺)，每週5天，持續十二週。結果發現，在給予運動訓練後，運動組的檸檬酸合成酵素活性明顯較控制組高，而體重明顯較控制組低。由oil red-O染色的結果顯示，運動訓練可降低胸主動脈竇上脂肪堆積的情形達18%，但不影響血清中總膽固醇和低密度脂蛋白膽固醇的含量。利用dihydroethidium的螢光染色可知，運動訓練後，運動組和控制組血管上超氧化物的表現沒有差異。以西方墨點雜合法測蛋白表現量，發現長期的運動訓練對血管細胞Bcl-2、超氧歧化酵素和熱休克蛋白72的表現沒有影響。綜合以上的結果可知，長期的運動訓練可以延緩動脈粥狀硬化的情形，且這可能和細胞凋亡相關蛋白的表現無直接的關係。

　Atherosclerosis is the leading cause of mortality in modern society. It has been reported that chronic exercise is an anti-atherosclerotic therapy. Previous studies have demonstrated that chronic exercise training reduces the apoptosis and the expression of pro-apoptotic proteins in skeletal muscle cells. It is also noticed that apoptotic events are involved in atherosclerotic progression. Whether exercise training reduces apoptosis of vascular cells in atherosclerosis remains unknown. The objective of this study was to investigate the effects of chronic exercise training on the expression of the apoptosis-related protein in atherosclerotic aorta. Male ApoE-/- mice fed with 0.15% cholesterol diet were trained on a treadmill for 60 minutes twice per day (at speed of 15m/min), 5 days per week for 12 weeks. After exercise training, the citrate synthase activity was higher and the body weight was lower in the exercise group than in the control group. Results of oil red-O staining showed that exercise training reduced the fat deposits up to 18%. However, there were no significant differences in serum total cholesterol and low density lipoprotein cholesterol levels. By detecting the dihydroethidium fluorescence, the superoxide production between two groups was no difference. The expressions of the apoptotic regulating proteins were further determined by Western blotting. After exercise training, there were no differences in the expression of Bcl-2, Cu/Zn SOD and Hsp72 protein. In conclusion, chronic exercise training reduces the progression of atherosclerosis. This reduction may not relate to expression of apoptosis-related proteins.

目錄
圖錄 v
表錄 vi
中文摘要 1
Abstract 2
緒論 4
(一) 動脈粥狀硬化與細胞凋亡的關係 4
(二) 細胞凋亡 4
(三) 氧化壓力 7
(四) 超氧歧化酵素 (superoxide dismutase,SOD) 8
(五) 熱休克蛋白質72 (heat shock protein 72,Hsp72) 9
(六) 運動 10
材料與方法 12
實驗動物 12
實驗動物的基因定型分析 (Genotyping) 12
高膽固醇飼料餵食 13
慢性中度運動 (Chronic Moderate Exercise) 13
動物犠牲 (Sacrifice)及組織取樣 13
血膽固醇之測定 15
冷凍切片 15
超氧化物的檢測 16
血管切片的脂肪堆積染色與量化 17
檸檬酸合成酵素活性 (Citrate Synthase Activity) 之檢測 17
蛋白質之萃取 19
蛋白質濃度之測定 19
蛋白質電泳與西方墨點雜合法 20
製備SDS-PAGE 20
電泳 (electrophoresis) 21
蛋白質電泳轉漬 (transfer) 21
雜交與偵測 (hybridization and detection) 21
統計分析 22
結果 23
基因定型分析結果 23
運動訓練後體重之變化 23
運動訓練後比目魚肌內檸檬酸合成酵素活性之變化 24
運動訓練後血清中血脂濃度之變化 24
運動訓練後血管上脂肪堆積之變化 25
運動訓練後血管上超氧化物表現之變化 25
運動訓練後血管上細胞凋亡相關蛋白表現之變化 25
討論 26
附錄 39
Krebs-Ringer-Hepes solution 39
6X Sample buffer 39
10X Running buffer 40
10X Transfer buffer 40
1X Transfer buffer 40
10X PBS 40
1X PBS-T 41
參考文獻 42

Beere, H. M., Wolf, B. B., Cain, K., Mosser, D. D., Mahboubi, A., Kuwana, T., Tailor, P., Morimoto, R. I., Cohen, G. M., and Green, D. R. (2000). Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome. Nat Cell Biol 2, 469-475.
Brandes, R. P., and Kreuzer, J. (2005). Vascular NADPH oxidases: molecular mechanisms of activation. Cardiovasc Res 65, 16-27.
Buttery, L. D., Springall, D. R., Chester, A. H., Evans, T. J., Standfield, E. N., Parums, D. V., Yacoub, M. H., and Polak, J. M. (1996). Inducible nitric oxide synthase is present within human atherosclerotic lesions and promotes the formation and activity of peroxynitrite. Lab Invest 75, 77-85.
Cai, H., and Harrison, D. G. (2000). Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res 87, 840-844.
Chen, K., Thomas, S. R., and Keaney, J. F., Jr. (2003). Beyond LDL oxidation: ROS in vascular signal transduction. Free Radic Biol Med 35, 117-132.
Chu, T. F., Huang, T. Y., Jen, C. J., and Chen, H. I. (2000). Effects of chronic exercise on calcium signaling in rat vascular endothelium. Am J Physiol Heart Circ Physiol 279, H1441-1446.
Cromheeke, K. M., Kockx, M. M., De Meyer, G. R., Bosmans, J. M., Bult, H., Beelaerts, W. J., Vrints, C. J., and Herman, A. G. (1999). Inducible nitric oxide synthase colocalizes with signs of lipid oxidation/peroxidation in human atherosclerotic plaques. Cardiovasc Res 43, 744-754.
Daiber, A., August, M., Baldus, S., Wendt, M., Oelze, M., Sydow, K., Kleschyov, A. L., and Munzel, T. (2004). Measurement of NAD(P)H oxidase-derived superoxide with the luminol analogue L-012. Free Radic Biol Med 36, 101-111.
De Keulenaer, G. W., Chappell, D. C., Ishizaka, N., Nerem, R. M., Alexander, R. W., and Griendling, K. K. (1998). Oscillatory and steady laminar shear stress differentially affect human endothelial redox state: role of a superoxide-producing NADH oxidase. Circ Res 82, 1094-1101.
de Nigris, F., Lerman, A., Ignarro, L. J., Williams-Ignarro, S., Sica, V., Baker, A. H., Lerman, L. O., Geng, Y. J., and Napoli, C. (2003). Oxidation-sensitive mechanisms, vascular apoptosis and atherosclerosis. Trends Mol Med 9, 351-359.
Degterev, A., Boyce, M., and Yuan, J. (2003). A decade of caspases. Oncogene 22, 8543-8567.
Dimmeler, S., Assmus, B., Hermann, C., Haendeler, J., and Zeiher, A. M. (1998). Fluid shear stress stimulates phosphorylation of Akt in human endothelial cells: involvement in suppression of apoptosis. Circ Res 83, 334-341.
Dimmeler, S., Haendeler, J., Galle, J., and Zeiher, A. M. (1997a). Oxidized low-density lipoprotein induces apoptosis of human endothelial cells by activation of CPP32-like proteases. A mechanistic clue to the 'response to injury' hypothesis. Circulation 95, 1760-1763.
Dimmeler, S., Haendeler, J., Rippmann, V., Nehls, M., and Zeiher, A. M. (1996). Shear stress inhibits apoptosis of human endothelial cells. FEBS Lett 399, 71-74.
Dimmeler, S., Rippmann, V., Weiland, U., Haendeler, J., and Zeiher, A. M. (1997b). Angiotensin II induces apoptosis of human endothelial cells. Protective effect of nitric oxide. Circ Res 81, 970-976.
Dimmeler, S., and Zeiher, A. M. (2003). Exercise and cardiovascular health: get active to "AKTivate" your endothelial nitric oxide synthase. Circulation 107, 3118-3120.
Du, X. L., Sui, G. Z., Stockklauser-Farber, K., Weiss, J., Zink, S., Schwippert, B., Wu, Q. X., Tschope, D., and Rosen, P. (1998). Introduction of apoptosis by high proinsulin and glucose in cultured human umbilical vein endothelial cells is mediated by reactive oxygen species. Diabetologia 41, 249-256.
Faraci, F. M., and Didion, S. P. (2004). Vascular protection: superoxide dismutase isoforms in the vessel wall. Arterioscler Thromb Vasc Biol 24, 1367-1373.
Finkel, T. (2003). Oxidant signals and oxidative stress. Curr Opin Cell Biol 15, 247-254.
Fukai, T., Siegfried, M. R., Ushio-Fukai, M., Cheng, Y., Kojda, G., and Harrison, D. G. (2000). Regulation of the vascular extracellular superoxide dismutase by nitric oxide and exercise training. J Clin Invest 105, 1631-1639.
Galis, Z. S., Sukhova, G. K., Lark, M. W., and Libby, P. (1994). Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest 94, 2493-2503.
Guo, Z., Van Remmen, H., Yang, H., Chen, X., Mele, J., Vijg, J., Epstein, C. J., Ho, Y. S., and Richardson, A. (2001). Changes in expression of antioxidant enzymes affect cell-mediated LDL oxidation and oxidized LDL-induced apoptosis in mouse aortic cells. Arterioscler Thromb Vasc Biol 21, 1131-1138.
Isner, J. M., Kearney, M., Bortman, S., and Passeri, J. (1995). Apoptosis in human atherosclerosis and restenosis. Circulation 91, 2703-2711.
Jen, C. J., Chan, H. P., and Chen, H. I. (2002). Chronic exercise improves endothelial calcium signaling and vasodilatation in hypercholesterolemic rabbit femoral artery. Arterioscler Thromb Vasc Biol 22, 1219-1224.
Kanwar, R. K., Kanwar, J. R., Wang, D., Ormrod, D. J., and Krissansen, G. W. (2001). Temporal expression of heat shock proteins 60 and 70 at lesion-prone sites during atherogenesis in ApoE-deficient mice. Arterioscler Thromb Vasc Biol 21, 1991-1997.
Kennedy, S. L., Smith, T. P., and Fleshner, M. (2005). Resting cellular and physiological effects of freewheel running. Med Sci Sports Exerc 37, 79-83.
Kockx, M. M., De Meyer, G. R., Muhring, J., Bult, H., Bultinck, J., and Herman, A. G. (1996). Distribution of cell replication and apoptosis in atherosclerotic plaques of cholesterol-fed rabbits. Atherosclerosis 120, 115-124.
Kockx, M. M., De Meyer, G. R., Muhring, J., Jacob, W., Bult, H., and Herman, A. G. (1998). Apoptosis and related proteins in different stages of human atherosclerotic plaques. Circulation 97, 2307-2315.
Kockx, M. M., and Herman, A. G. (2000). Apoptosis in atherosclerosis: beneficial or detrimental? Cardiovasc Res 45, 736-746.
Kuwana, T., and Newmeyer, D. D. (2003). Bcl-2-family proteins and the role of mitochondria in apoptosis. Curr Opin Cell Biol 15, 691-699.
Laufs, U., Wassmann, S., Czech, T., Munzel, T., Eisenhauer, M., Bohm, M., and Nickenig, G. (2005). Physical inactivity increases oxidative stress, endothelial dysfunction, and atherosclerosis. Arterioscler Thromb Vasc Biol 25, 809-814.
Lizard, G., Lemaire, S., Monier, S., Gueldry, S., Neel, D., and Gambert, P. (1997). Induction of apoptosis and of interleukin-1beta secretion by 7beta-hydroxycholesterol and 7-ketocholesterol: partial inhibition by Bcl-2 overexpression. FEBS Lett 419, 276-280.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265-275.
Luoma, J. S., Stralin, P., Marklund, S. L., Hiltunen, T. P., Sarkioja, T., and Yla-Herttuala, S. (1998). Expression of extracellular SOD and iNOS in macrophages and smooth muscle cells in human and rabbit atherosclerotic lesions: colocalization with epitopes characteristic of oxidized LDL and peroxynitrite-modified proteins. Arterioscler Thromb Vasc Biol 18, 157-167.
Madamanchi, N. R., Vendrov, A., and Runge, M. S. (2005). Oxidative stress and vascular disease. Arterioscler Thromb Vasc Biol 25, 29-38.
Marumo, T., Schini-Kerth, V. B., Fisslthaler, B., and Busse, R. (1997). Platelet-derived growth factor-stimulated superoxide anion production modulates activation of transcription factor NF-kappaB and expression of monocyte chemoattractant protein 1 in human aortic smooth muscle cells. Circulation 96, 2361-2367.
Mehta, T. A., Greenman, J., Ettelaie, C., Venkatasubramaniam, A., Chetter, I. C., and McCollum, P. T. (2005). Heat shock proteins in vascular disease--a review. Eur J Vasc Endovasc Surg 29, 395-402.
Meilhac, O., Ramachandran, S., Chiang, K., Santanam, N., and Parthasarathy, S. (2001). Role of arterial wall antioxidant defense in beneficial effects of exercise on atherosclerosis in mice. Arterioscler Thromb Vasc Biol 21, 1681-1688.
Napoli, C., Williams-Ignarro, S., De Nigris, F., Lerman, L. O., Rossi, L., Guarino, C., Mansueto, G., Di Tuoro, F., Pignalosa, O., De Rosa, G., et al. (2004). Long-term combined beneficial effects of physical training and metabolic treatment on atherosclerosis in hypercholesterolemic mice. Proc Natl Acad Sci U S A 101, 8797-8802.
Newby, A. C., and Zaltsman, A. B. (1999). Fibrous cap formation or destruction--the critical importance of vascular smooth muscle cell proliferation, migration and matrix formation. Cardiovasc Res 41, 345-360.
Okada, M., Hasebe, N., Aizawa, Y., Izawa, K., Kawabe, J., and Kikuchi, K. (2004). Thermal treatment attenuates neointimal thickening with enhanced expression of heat-shock protein 72 and suppression of oxidative stress. Circulation 109, 1763-1768.
Ross, R. (1999). Atherosclerosis--an inflammatory disease. N Engl J Med 340, 115-126.
Shephard, R. J., and Balady, G. J. (1999). Exercise as cardiovascular therapy. Circulation 99, 963-972.
Siu, P. M., Bryner, R. W., Martyn, J. K., and Alway, S. E. (2004). Apoptotic adaptations from exercise training in skeletal and cardiac muscles. FASEB J 18, 1150-1152.
Srere, P. A (1969). Citrate synthase. Methods Enzymol 13, 3-5.
Tarpey, M. M., and Fridovich, I. (2001). Methods of detection of vascular reactive species: nitric oxide, superoxide, hydrogen peroxide, and peroxynitrite. Circ Res 89, 224-236.
Ueda, H., and Fujita, R. (2004). Cell death mode switch from necrosis to apoptosis in brain. Biol Pharm Bull 27, 950-955.
Valgimigli, M., Merli, E., Malagutti, P., Soukhomovskaia, O., Cicchitelli, G., Macri, G., and Ferrari, R. (2003). Endothelial dysfunction in acute and chronic coronary syndromes: evidence for a pathogenetic role of oxidative stress. Arch Biochem Biophys 420, 255-261.
van der Wal, A. C., and Becker, A. E. (1999). Atherosclerotic plaque rupture--pathologic basis of plaque stability and instability. Cardiovasc Res 41, 334-344.
van Eden, W., van der Zee, R., and Prakken, B. (2005). Heat-shock proteins induce T-cell regulation of chronic inflammation. Nat Rev Immunol 5, 318-330.
van Loo, G., Saelens, X., van Gurp, M., MacFarlane, M., Martin, S. J., and Vandenabeele, P. (2002). The role of mitochondrial factors in apoptosis: a Russian roulette with more than one bullet. Cell Death Differ 9, 1031-1042.
Wick, G., Perschinka, H., and Millonig, G. (2001). Atherosclerosis as an autoimmune disease: an update. Trends Immunol 22, 665-669.
Wilcox, J. N., Subramanian, R. R., Sundell, C. L., Tracey, W. R., Pollock, J. S., Harrison, D. G., and Marsden, P. A. (1997). Expression of multiple isoforms of nitric oxide synthase in normal and atherosclerotic vessels. Arterioscler Thromb Vasc Biol 17, 2479-2488.
Xu, Q. (2003). Infections, heat shock proteins, and atherosclerosis. Curr Opin Cardiol 18, 245-252.
Yang, A. L., and Chen, H. I. (2003). Chronic exercise reduces adhesion molecules/iNOS expression and partially reverses vascular responsiveness in hypercholesterolemic rabbit aortae. Atherosclerosis 169, 11-17.