臺灣博碩士論文加值系統

血管內皮前驅細胞 ( endothelial progenitor cells；EPCs ) 其具有血管新生的作用，相關的研究顯示其能改善缺血性傷害，缺血性傷害發生於許多疾病當中，針對糖尿病來說，在高血糖的情況下會使血中的 EPCs產生衰老現象，進而減少 EPCs 的數量和損害其功能，使得側枝血管減少最終造成缺血性傷害，魚油包含了 eicosapentaenoic acid ( EPA;20:5n-3 )和 docosahexaenoic acid ( DHA; 22:6n-3 )，目前有許多研究已指出飲食中多攝取魚油可有效降低罹患心血管疾病、中風的風險，魚油被認為有預防缺血性傷害的功能，所以本研究目的在探討魚油對於改善糖尿病缺血性傷害的機制。

Endothelial progenitor cells (EPCs) play a significant role in postnatal neovascularization. There are many studies demonstrated that EPCs could improve the ischemic injury. Ischemic injury which occurs in many diseases, for diabetes, high glucose may enhance cellular senescence and decrease cell numbers and functional competencies of EPCs. Fish oil contain the long-chain polyunsaturated omega-3 fatty acids eicosapentaenoic acid (EPA: 20:5n-3) and docosahexaenoic acid (DHA: 22:6n-3). There are many studies demonstrated that fish oil have the benefits on the prevention of the cardiovascular disease and stroke. Recently it was demonstrated that fish oil have the benefits on the prevention of the ischemic disease.

目錄 i
圖目錄 iv
摘要 v
Abstract vi
第一章、前言 1
第二章、文獻探討 3
一、 血管內皮前驅細胞( Endothelial progenitor cells ) 3
(1)來源、功能 3
(2)血管內皮前驅細胞與缺血性傷害 4
二、 糖尿病 ( Diabetes ) 5
(1) 定義與分類 5
(2) 糖尿病與缺血性傷害 6
三、 魚油 ( Fish oil ) 7
(1) 生理功能 7
(2) 魚油的生理濃度及建議攝取量 8
(3) 魚油與糖尿病缺血性傷害 9
四、 與EPCs細胞相關血管生成蛋白質 10
(1) c -kit 10
(2) Vascular endothelial-cadherin ( VE-cadherin ) 10
(3) Endothelial nitric-oxide synthase (eNOS) 11
五、 細胞週期相關蛋白 11
六、 c-Myc 12
七、 MAPK/ERK訊息傳遞路 12
八、 AMPK訊息傳遞路徑 13
九、 實驗目的與動機 13
第三章、材料與方法 15
一、 實驗架構 15
二、 實驗藥品與耗材 17
三、 細胞來源 18
四、 細胞培養 (Cell culture) 18
五、 細胞存活率分析 (MTT assay) 20
六、 血管形成試驗 (Tube formation assay) 21
七、 總蛋白質萃取 21
八、 細胞質、核蛋白萃取 (NE-PER Nuclear and Cytoplasmic Extraction
Reagents) 22
九、 蛋白質定量 23
十、 西方點墨法(western blot) 23
十一、 實驗動物 25
十二、 老鼠下肢缺血模式(Mouse hind limb ischemia modal) 26
十三、 都卜勒血流影像系統(Laser Doppler perfusion imager system) 27
十四、 統計方法 28
第四章、結果 29
一、 高葡萄糖對於細胞存活率的影響 29
二、 高葡萄糖對於細胞血管形成能力的影響 31
三、 在高葡萄糖下EPA 以及 DHA對於血管形成能力的影響 34
四、 DHA 可能藉由MAPK/ERK以及AMPK pathway 調控高葡萄糖下
細胞血管形成能力 37
五、 在高葡萄糖下 DHA 具有影響血管生成蛋白質表現的能力 39
六、 在高葡萄糖下 DHA 具有調控細胞中 MAPK/ERK以及AMPK
pathway 的能力 42
七、 在高葡萄糖下 DHA具有影響細胞週期相關蛋白表現的能力 45
八、 在下肢缺血動物模式中魚油的攝取可以增進血管新生來幫助側枝血
流的復原 47
第五章、討論 49
第六章、結論 53
第七章、參考文獻 54
第八章、附錄 59

1.Kawamoto, A., et al., Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation, 2001. 103(5): p. 634-7.
2.Assmus, B., et al., Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI). Circulation, 2002. 106(24): p. 3009-17.
3.Kalka, C., et al., Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci U S A, 2000. 97(7): p. 3422-7.
4.衛生福利部。糖尿病之流行病學及病因、診斷、分類.
5.Tepper, O.M., et al., Human endothelial progenitor cells from type II diabetics exhibit impaired proliferation, adhesion, and incorporation into vascular structures. Circulation, 2002. 106(22): p. 2781-6.
6.Lavie, C.J., et al., Omega-3 polyunsaturated fatty acids and cardiovascular diseases. J Am Coll Cardiol, 2009. 54(7): p. 585-94.
7.Asahara, T., et al., Isolation of putative progenitor endothelial cells for angiogenesis. Science, 1997. 275(5302): p. 964-7.
8.Bauer, S.M., et al., The bone marrow-derived endothelial progenitor cell response is impaired in delayed wound healing from ischemia. J Vasc Surg, 2006. 43(1): p. 134-41.
9.Murohara, T., et al., Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J Clin Invest, 2000. 105(11): p. 1527-36.
10.Schmidt, A., K. Brixius, and W. Bloch, Endothelial precursor cell migration during vasculogenesis. Circ Res, 2007. 101(2): p. 125-36.
11.Tateishi-Yuyama, E., et al., Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial. Lancet, 2002. 360(9331): p. 427-35.
12.Classification and diagnosis of diabetes mellitus and other categories of glucose intolerance. National Diabetes Data Group. Diabetes, 1979. 28(12): p. 1039-57.
13.Sandbaek, A., et al., Effect of Early Multifactorial Therapy Compared With Routine Care on Microvascular Outcomes at 5 Years in People With Screen-Detected Diabetes: A Randomised Controlled Trial: The ADDITION-Europe Study. Diabetes Care, 2014.
14.Creager, M.A., et al., Diabetes and vascular disease: pathophysiology, clinical consequences, and medical therapy: Part I. Circulation, 2003. 108(12): p. 1527-32.
15.Gupta, A.K., et al., Endothelial Dysfunction: An Early Cardiovascular Risk Marker in Asymptomatic Obese Individuals with Prediabetes. Br J Med Med Res, 2012. 2(3): p. 413-423.
16.Ruiter, M.S., et al., Diabetes impairs arteriogenesis in the peripheral circulation: review of molecular mechanisms. Clin Sci (Lond), 2010. 119(6): p. 225-38.
17.Chen, Y.H., et al., High glucose impairs early and late endothelial progenitor cells by modifying nitric oxide-related but not oxidative stress-mediated mechanisms. Diabetes, 2007. 56(6): p. 1559-68.
18.Kang, L., et al., Decreased mobilization of endothelial progenitor cells contributes to impaired neovascularization in diabetes. Clin Exp Pharmacol Physiol, 2009. 36(10): p. e47-56.
19.Bang, H.O., J. Dyerberg, and A.B. Nielsen, Plasma lipid and lipoprotein pattern in Greenlandic West-coast Eskimos. Lancet, 1971. 1(7710): p. 1143-5.
20.Calder, P.C., n-3 polyunsaturated fatty acids, inflammation, and inflammatory diseases. Am J Clin Nutr, 2006. 83(6 Suppl): p. 1505S-1519S.
21.Massaro, M., et al., Basic mechanisms behind the effects of n-3 fatty acids on cardiovascular disease. Prostaglandins Leukot Essent Fatty Acids, 2008. 79(3-5): p. 109-15.
22.Goodnight, S.H., Jr., et al., Polyunsaturated fatty acids, hyperlipidemia, and thrombosis. Arteriosclerosis, 1982. 2(2): p. 87-113.
23.Higgs, G.A., et al., The source of thromboxane and prostaglandins in experimental inflammation. Br J Pharmacol, 1983. 79(4): p. 863-8.
24.Harper, C.R., et al., Flaxseed oil increases the plasma concentrations of cardioprotective (n-3) fatty acids in humans. J Nutr, 2006. 136(1): p. 83-7.
25.Welch, A.A., et al., Dietary fish intake and plasma phospholipid n-3 polyunsaturated fatty acid concentrations in men and women in the European Prospective Investigation into Cancer-Norfolk United Kingdom cohort. Am J Clin Nutr, 2006. 84(6): p. 1330-9.
26.Rosell, M.S., et al., Long-chain n-3 polyunsaturated fatty acids in plasma in British meat-eating, vegetarian, and vegan men. Am J Clin Nutr, 2005. 82(2): p. 327-34.
27.Kris-Etherton, P.M., W.S. Harris, and L.J. Appel, Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease. Circulation, 2002. 106(21): p. 2747-57.
28.He, K., et al., Fish consumption and incidence of stroke: a meta-analysis of cohort studies. Stroke, 2004. 35(7): p. 1538-42.
29.Losordo, D.W. and S. Dimmeler, Therapeutic angiogenesis and vasculogenesis for ischemic disease: part II: cell-based therapies. Circulation, 2004. 109(22): p. 2692-7.
30.Burger, D. and R.M. Touyz, Cellular biomarkers of endothelial health: microparticles, endothelial progenitor cells, and circulating endothelial cells. J Am Soc Hypertens, 2012. 6(2): p. 85-99.
31.Devaraj, S., et al., Modulation of endothelial progenitor cell number and function with n-3 polyunsaturated fatty acids. Atherosclerosis, 2013. 228(1): p. 94-7.
32.Turgeon, J., et al., Fish oil-enriched diet protects against ischemia by improving angiogenesis, endothelial progenitor cell function and postnatal neovascularization. Atherosclerosis, 2013. 229(2): p. 295-303.
33.Tikhonenko, M., et al., N-3 polyunsaturated Fatty acids prevent diabetic retinopathy by inhibition of retinal vascular damage and enhanced endothelial progenitor cell reparative function. PLoS One, 2013. 8(1): p. e55177.
34.Martin, F.H., et al., Primary structure and functional expression of rat and human stem cell factor DNAs. Cell, 1990. 63(1): p. 203-11.
35.Gommerman, J.L., R. Rottapel, and S.A. Berger, Phosphatidylinositol 3-kinase and Ca2+ influx dependence for ligand-stimulated internalization of the c-Kit receptor. J Biol Chem, 1997. 272(48): p. 30519-25.
36.Dentelli, P., et al., C-KIT, by interacting with the membrane-bound ligand, recruits endothelial progenitor cells to inflamed endothelium. Blood, 2007. 109(10): p. 4264-71.
37.Matsui, J., et al., Stem cell factor/c-kit signaling promotes the survival, migration, and capillary tube formation of human umbilical vein endothelial cells. J Biol Chem, 2004. 279(18): p. 18600-7.
38.Kapur, R., et al., Role of p38 and ERK MAP kinase in proliferation of erythroid progenitors in response to stimulation by soluble and membrane isoforms of stem cell factor. Blood, 2002. 100(4): p. 1287-93.
39.Gumbiner, B.M., Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell, 1996. 84(3): p. 345-57.
40.Lim, W.H., et al., Stent coated with antibody against vascular endothelial-cadherin captures endothelial progenitor cells, accelerates re-endothelialization, and reduces neointimal formation. Arterioscler Thromb Vasc Biol, 2011. 31(12): p. 2798-805.
41.Yamamoto, K., et al., Proliferation, differentiation, and tube formation by endothelial progenitor cells in response to shear stress. J Appl Physiol (1985), 2003. 95(5): p. 2081-8.
42.Fulton, D., J.P. Gratton, and W.C. Sessa, Post-translational control of endothelial nitric oxide synthase: why isn''t calcium/calmodulin enough? J Pharmacol Exp Ther, 2001. 299(3): p. 818-24.
43.Shaul, P.W., Regulation of endothelial nitric oxide synthase: location, location, location. Annu Rev Physiol, 2002. 64: p. 749-74.
44.Aicher, A., et al., Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells. Nat Med, 2003. 9(11): p. 1370-6.
45.Thum, T., et al., Endothelial nitric oxide synthase uncoupling impairs endothelial progenitor cell mobilization and function in diabetes. Diabetes, 2007. 56(3): p. 666-74.
46.Nurse, P., Y. Masui, and L. Hartwell, Understanding the cell cycle. Nat Med, 1998. 4(10): p. 1103-6.
47.Nurse, P.M., Nobel Lecture. Cyclin dependent kinases and cell cycle control. Biosci Rep, 2002. 22(5-6): p. 487-99.
48.Sherr, C.J. and J.M. Roberts, Living with or without cyclins and cyclin-dependent kinases. Genes Dev, 2004. 18(22): p. 2699-711.
49.Blackwood, E.M. and R.N. Eisenman, Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science, 1991. 251(4998): p. 1211-7.
50.Baudino, T.A. and J.L. Cleveland, The Max network gone mad. Mol Cell Biol, 2001. 21(3): p. 691-702.
51.Pearson, G., et al., Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev, 2001. 22(2): p. 153-83.
52.Carling, D., et al., AMP-activated protein kinase: nature''s energy sensor. Nat Chem Biol, 2011. 7(8): p. 512-8.
53.Hardie, D.G., AMP-activated protein kinase: an energy sensor that regulates all aspects of cell function. Genes Dev, 2011. 25(18): p. 1895-908.
54.Zhang, B.B., G. Zhou, and C. Li, AMPK: an emerging drug target for diabetes and the metabolic syndrome. Cell Metab, 2009. 9(5): p. 407-16.
55.Xiang, L., et al., Chronic hyperglycemia impairs functional vasodilation via increasing thromboxane-receptor-mediated vasoconstriction. Am J Physiol Heart Circ Physiol, 2007. 292(1): p. H231-6.
56.Gaenzer, H., et al., Effect of insulin therapy on endothelium-dependent dilation in type 2 diabetes mellitus. Am J Cardiol, 2002. 89(4): p. 431-4.
57.Chiu, S.C., et al., Eicosapentaenoic acid induces neovasculogenesis in human endothelial progenitor cells by modulating c-kit protein and PI3-K/Akt/eNOS signaling pathways. J Nutr Biochem, 2014. 25(9): p. 934-45.
58.Jiraritthamrong, C., et al., In vitro vessel-forming capacity of endothelial progenitor cells in high glucose conditions. Ann Hematol, 2012. 91(3): p. 311-20.
59.Musi, N., et al., Metformin increases AMP-activated protein kinase activity in skeletal muscle of subjects with type 2 diabetes. Diabetes, 2002. 51(7): p. 2074-81.
60.Li, X., et al., AMP-activated protein kinase promotes the differentiation of endothelial progenitor cells. Arterioscler Thromb Vasc Biol, 2008. 28(10): p. 1789-95.