臺灣博碩士論文加值系統

心血管疾病導致的心臟衰竭、心肌梗塞和中風是人類死亡的主要原因，而心血管疾病是由動脈硬化所引起，在動脈硬化斑中含有大量的組織因子 ( tissue factor, TF )，當動脈硬化斑破裂時，組織因子暴露在血管內，引發一連串的凝血反應，最後形成血栓而阻塞血管，是造成急性症狀及病人死亡的主要原因。過去的研究指出，β-glucan能降低血中膽固醇濃度，但是對組織因子的表現是否有相同效果則不明，不同來源的β-glucan作用可能不同，此實驗的目的是要瞭解不同來源的β-glucan 對組織因子表現的影響。本實驗餵食紐西蘭白兔高脂高膽固醇飼料，誘發高膽固醇血症造成硬化斑，作為動脈硬化的動物模型。首先測試餵食不同配方飼料的效果，以LPS ( lipopolysaccharide ) 刺激周邊血液單核球 ( peripheral blood mononuclear cells, PBMC ) 後，測量其組織因子的活性，結果顯示，餵食六週1% 膽固醇加5%豬油或10%豬油這兩組，其組織因子活性有顯著的升高。試驗二，餵食兔子1% 膽固醇加5%豬油，同時添加自酵母萃取的β-glucan ( 0.1% ~ 0.3%)，研究其對組織因子活性的影響，餵食四週、六週後，測量其周邊血液單核球及主動脈的組織因子活性，結果顯示，餵食酵母β-glucan的兔子其組織因子活性有降低的趨勢( p<0.1 )。試驗三，比較不同來源的β-glucan的作用，兔子在餵食1% 膽固醇加5% 豬油誘發動脈硬化後，分別在飼料中添加酵母或大麥β-glucan，繼續餵食四週、六週後，測量其周邊血液單核球及主動脈的組織因子活性，以及動脈組織因子mRNA表現量，比較0.3%的酵母β(1-3, 1-6)-glucan和0.3%、0.5% 大麥β(1-3, 1-4)-glucan的效果，結果顯示餵食大麥β-glucan六週後，組織因子活性有顯著的下降( p <0.05 )，RT-PCR的結果也顯示組織因子 mRNA表現量有顯著的下降。根據本實驗的結果顯示，食用 β-glucan 除了可以降低血中膽固醇濃度，也可降低組織因子的表現量，而大麥的 β(1-3, 1-4)-glucan 比酵母的 β(1-3, 1-6)-glucan 能更有效的降低組織因子的表現。

Cardiovascular disease including chronic heart failure, myocardial infarction, and stroke is the major cause of morbidity and mortality. Atherosclerosis is major contributor to cardiovascular disease. Triggering of thrombosis by plaque disruption is probably the most important mechanisms leading to the onset of acute arterial disease and ischemic sudden death. Tissue factor (TF) is highly expressed in atherosclerotic plaques, and its content has been related to plaque thrombogenicity. β-glucan is a polysaccharide and the main content of fiber in grains, yeast and mushrooms. Recent studies showed β-glucan have various effects including lipid lowering, and β-glucan from different source have different biological activity. In this study, we attempt to investigate the effect of β-glucan from different source on the TF expression. The New Zealand White rabbits feeding a high cholesterol, high lipid diet were used as an animal model. At first, diet contain 1% cholesterol only or plus different concentration lard oil ( 5% and 10%) were tested for hypercholesterolemia induction and TF activity analysis. Peripheral blood mononuclear cells (PBMC) were isolated and stimulated by lipopolysaccharide (LPS), then the TF activity were measured by chromogenic assay or clotting time. Increacing TF activity was observed in all rabbits fed cholesterol and cholesterol plus lard oil. The two groups fed cholesterol plus lard oil had the highest level of TF activity after six weeks of feeding. In test 2, we analyzed the effct of yeast β-glucan. Different concentartion of yeast β-glucan (0.1, 0.2 and 0.3%) were added into HCL diet (1% cholesterol plus 5% lard oil) for six weeks. The TF activity of PBMC and aorta were measured at four and six weeks. Compared to HCL diet only, rabbit fed β-glucan showed a trend of lower TF activity, but did not significant (p<0.1). In test 3, we compared the effect of different β-glucan. Rabbits assigned HCL diet (1% cholesterol plus 5% lard oil) to induce atherosclerosis, followed by HCL diet plus 0.3% yeast β(1-3, 1-6)-glucan, 0.3% or 0.5% barley β(1-3, 1-4)-glucan for six weeks. After 4 weeks and 6 weeks intake of β-glucan, TF activity of PBMC and aorta were measured, and the level of TF mRNA on aorta was determined by reverse - transcription polymerase chain reaction (RT-PCR). Both TF activity and TF mRNA expression showed significant decrease in barley β-glucan group ( p < 0.05 ), but not in yeast β-glucan group. These results suggest that intake of β-glucan could reduce expression and activity of TF. The barley β(1-3, 1-4)-glucan has more pronounced effect than yeast β(1-3, 1-6)-glucan on TF expression.

目錄..................................................I
圖目錄................................................IV
縮寫表................................................VI
中文摘要..............................................1
Abstract..............................................3
第一章、導論..........................................6
第二章、前言..........................................8
一、動脈粥狀硬化......................................8
二、動脈硬化與組織因子 ( TF )的關係...................9
(一) 功能.............................................9
(二) 結構............................................11
(三) TF 表現的位置...................................12
(四) TF 與動脈硬化...................................12
三、血纖維蛋白原與D-Dimer............................14
四、β-葡聚糖 ( β- glucan ............................15
(一) 特性............................................15
(二) 結構............................................15
(1) 酵母 β-glucan....................................15
(2) 穀類 β-glucan....................................16
(三) 生理功能........................................17
(1) 酵母β-glucan.................................... 17
(2) 穀類 β-glucan....................................18
四、本論文的目的.....................................19
第三章、材料及方法...................................20
一、動物實驗設計.....................................20
(一) 試驗一：建立紐西蘭白兔動脈硬化動物模式..........20
(二) 試驗二：飼料添加酵母β-glucan對紐西蘭兔動脈硬化之影響 ............................................20
(三) 試驗三：飼料添加不同β-glucan對紐西蘭兔動脈硬化之影響 ............................................21
(四) 飼料管理........................................21
二、分析方法.........................................22
(一) 血中膽固醇濃度分析..............................22
(二) 動脈免疫組織染色................................22
(三) 血漿凝血活性分析................................22
(1) Prothrombin time ( PT )..........................22
(2) Activated partial thromboplastin time ( aPTT )...22
(3) Thrombin time ( TT ).............................23
(4) 血纖維蛋白原( Fibrinogen ).......................23
(四) 組織因子活性分析................................24
(1) 週邊血液單核球分離及培養.........................24
(2) 週邊血液單核球組織因子之測定.....................24
(3) 動脈組織因子之測定...............................26
(五) 組織因子 mRNA 表現的測定........................27
(1) 動脈 RNA 的萃取..................................27
(2) Reverse transcriptase polymerase chain reaction( RT - PCR )................................................28
三、統計分析.........................................29
第四章、實驗結果.....................................30
一、建立紐西蘭白兔動脈硬化動物模式...................30
二、飼料添加酵母β-glucan對紐西蘭兔動脈硬化之影響.....31
三、飼料添加不同β-glucan對紐西蘭兔動脈硬化之影響.....32
第五章、討論.........................................34
參考文獻.............................................38
結果圖表.............................................50

1. Anetta U., Kathleen E. B. and Kenneth G. M., Statins and blood coagulation. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 287-294.
2. Alison D. S., Barrett J. R., Yujun J. Z., Israel F. C., John T. F., Maria R., Peter L. A. G., Yale N. and Mark B. T. Tissue factor is induced by monocyte Chemoattractant Protein-1 in human aortic smooth muscle and THP-1 cells. The Journal Of Biological Chemistry 1997; 272: 28568-28573.
3. Battilana P., Ornstein K., Minehira K., Schwarz JM, Acheson K., Schneiter P., Burri J., Je�韡uier E. and Tappy L. Original communication mechanisms of action of β-glucan in postprandial glucose metabolism in healthy men. European Journal of Clinical Nutr. 2001; 55: 327-333.
4. Benagiano M, D'Elios MM, Amedei A, Azzurri A, van der Zee R, Ciervo A, Rombol�� G, Romagnani S, Cassone A, Del Prete G. Human 60-kDa heat shock protein is a target autoantigen of T cells derived from atherosclerotic plaques. J Immunol. 2005; 174: 6509-17.
5. Bhattacharjee G, Ahamed J, Pedersen B, El-Sheikh A, Mackman N, Ruf W, Liu C, Edgington TS. Regulation of tissue factor--mediated initiation of the coagulation cascade by cell surface grp78. Arterioscler Thromb Vasc Biol. 2005; 25: 1737-43.
6. Catena C, Novello M, Lapenna R, Baroselli S, Colussi G, Nadalini E, Favret G, Cavarape A, Soardo G, Sechi LA. New risk factors for atherosclerosis in hypertension: focus on the prothrombotic state and lipoprotein(a). J Hypertens. 2005; 23: 1617-31.
7. Chen J, Seviour R. Medicinal importance of fungal beta-(1-->3), (1-->6)-glucans. Mycol Res. 2007; 6: 635-52.
8. Chi L, Gibson G, Peng YW, Bousley R, Brammer D, Rekhter M, Chen J, Leadley R. Characterization of a tissue factor/factor VIIa-dependent model of thrombosis in hypercholesterolemic rabbits. J Thromb Haemost. 2004; 2: 85-92.
9. Christoph S., Kai Z., Matthias Th. and Matthias H. Heat shock inhibits lipopolysaccharide-induced tissue factor activity in human whole blood. Thrombosis Journal 2007; 5: 13-18.
10. Conde I., Corie N. S., Perumal T., and Jose? A. L. Tissue- factor-bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation. Blood. 2005; 106: 1604-1611.
11. Cristiana C., Marileda N., Roberta L., Sara B., Gianluca C., Elisa N., Grazia F., Alessandro C., Giorgio S. and Leonardo A. S. New risk factors for atherosclerosis in hypertension: focus on the prothrombotic state and lipoprotein ( a ). Journal of Hypertension 2005; 23: 1617-1631.
12. Cummings JH, Englyst HN. Gastrointestinal effects of food carbohydrate. Am J Clin Nutr. 1995; 61: 938S-945S.
13. Dackiw A. P. B., Grinstein S., Brisseau G. F., Mcgilvray I. D., Nathens A. B., Mcguire J. A., Romanek R., Cheung P. Y. C. and Rotstein O. D. The role of tyrosine phosphorylation in lipopoly- saccharideand zymosan-induced procoagulant activity and tissue factor expression in macrophages. Infection And Immunity 1997; 65: 2362-2370.
14. Di Santo A, Mezzetti A, Napoleone E, Di Tommaso R, Donati MB, De Gaetano G, Lorenzet R. Resveratrol and quercetin down-regulate tissue factor expression by human stimulated vascular cells. J Thromb Haemost. 2003; 1: 1089-95.
15. Emmanuelle J., Thierry L. T., Isabelle S., Christophe Z., Eric V. B., Michael D. E., R�縵is B., Sophie S., Brigitte J. and Delphine C. Dietary lipid lowering modifies plaque phenotype in rabbit atheroma after angioplasty: a potential role of tissue factor. Circulation 2003; 108: 1740-1745.
16. Eva L., Taavo T. and Agneta S. Role of Platelet P-Selectin and CD40 Ligand in the Induction of Monocytic Tissue Factor Expression Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2322-2328.
17. Fan J, Shimoyamada H, Sun H, Marcovina S, Honda K, Watanabe T. Transgenic rabbits expressing human apolipoprotein(a) develop more extensive atherosclerotic lesions in response to a cholesterol-rich diet. Arterioscler Thromb Vasc Biol. 2001; 21: 88-94.
18. Fei H., Berliner J.A., Parhami F. and Drake T.A., Regulation of endothelial cell tissue factor expression by minimally oxidized LDL and lipopolysaccharide. Arterioscler. Thromb. Vasc. Biol. 1993; 13: 1711-1717.
19. Gray GM. Starch digestion and absorption in nonruminants. J Nutr. 1992; 122: 172-7.
20. Himber J, Wohlgensinger C, Roux S, Damico LA, Fallon JT, Kirchhofer D, Nemerson Y, Riederer MA. Inhibition of tissue factor limits the growth of venous thrombus in the rabbit. J Thromb Haemost. 2003; 1: 889-95.
21. Jim A., Michael J. H., Laveena S., Piers D., Leon M., Will G. J., Julia L. G., A. Richard K., Ming L., and Peter G. T. The cytoplasmic domain of tissue factor in macrophages augments cutaneous delayed-type hypersensitivity. Journal of Leukocyte Biology 2008; 83: 1-10.
22. Jonathan E., Colleen F., John M., Richard J. U. and Nigel M. Lipopolysaccharide induction of tissue factor expression in rabbits. Infection and Immunity.1999; 67: 2540-2546.
23. Karoly M., Sharon A., Russell D., Raymund M., Arnold H., Cynthia B., Georgia T. and Brian J. Parent monocyte tissue factor induction by lipopolysaccharide (LPS): dependence on LPS-binding protein and CD14, and inhibition by a recombinant fragment of bactericidal / permeability-increasing protein. The American Society of Hema. 1994; 83: 2516-252.
24. Kay R. M. Dietary fiber. Journal of Lipid Research 1982; 23: 221-242.
25. Keenan JM, Goulson M, Shamliyan T et al. The effects of concentrated barley β-glucan on blood lipids in a population of hypercholesterolemic men and women. Br. J. Nutr. 2007; 97: 1162-1168.
26. Kim SY, Song HJ, Lee YY, Cho KH, Roh YK. Biomedical issues of dietary fiber beta-glucan. J Korean Med Sci. 2006 ; 21: 781-9.
27. Kumagai T, Hoshi Y, Tsutsumi H, Ebina K, Yokota K. Inhibition of plasma coagulation through interaction between oxidized low-density lipoprotein and blood coagulation factor VIII. Biol Pharm Bull. 2005; 28: 952-6.
28. Li X, Wen Z, He X, He S. Effects of cinnamic acid on expression of tissue factor induced by TNFalpha in endothelial cells and its mechanisms. J Chin Med Assoc. 2006; 69: 207-12.
29. Lifschitz CH, Grusak MA, Butte NF. Carbohydrate digestion in humans from a beta-glucan-enriched barley is reduced. J Nutr. 2002; 132: 2593-6.
30. Lindmark E, Tenno T, Chen J, Siegbahn A. IL-10 inhibits LPS-induced human monocyte tissue factor expression in whole blood. Br J Haematol. 1998; 102: 597-604.
31. Liu M. L., Michael P. R., Peter C., Steven E. M. and Kevin J. W. Cholesterol enrichment of human monocyte / macrophages induces surface exposure of phosphatidylserine and the release of biologically-active tissue factor-positive microvesicles. Aterioscler. Thromb. Vasc. Biol. 2007;27;430-435
32. Lopez-Vilchez I, Escolar G, Diaz-Ricart M, Fuste B, Galan AM, White JG. Tissue factor-enriched vesicles are taken up by platelets and induce platelet aggregation in the presence of factor VIIa. Thromb Haemost. 2007;2: 202-211.
33. Lucy A. N., Sinead W., Anne N., Helen M. R. LPS induced tissue factor expression in the THP-1 monocyte cell line is attenuated by conjugated linoleic acid. Thromb. Res. 2006; 117: 475-480.
34. Mackman N. Role of tissue factor in hemostasis, thrombosis, and vascular development. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1015-1022.
35. Mark M. E. D., Jacqueline T., Leny H., Arnoud L. and Rogier M. B., Cholesterol or triglyceride loading of luman monocyte-derived macrophages by incubation with modified lipoproteins does not induce tissue factor expression. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 384-392.
36. Masanori A., Sami J. V., Seigo S., Elena R., Mark B. T., John T. F. and Peter L. Dietary lipid lowering reduces tissue factor expression in rabbit atheroma. Circulation 1999; 100: 1215-1222.
37. Mojca S., Nina V., Mojca B. Do haemostasis activation markers that predict cardiovascular disease exist. Pathophysiol Haemost Thromb 2004; 33: 302-308.
38. Mueller A, Raptis J, Rice PJ, Kalbfleisch JH, Stout RD, Ensley HE, Browder W, Williams DL. The influence of glucan polymer structure and solution conformation on binding to (1-->3)-beta-D-glucan receptors in a human monocyte-like cell line. Glycobiology. 2000; 10: 339-46.
39. M�刜ler I, Klocke A, Alex M, Kotzsch M, Luther T, Morgenstern E, Zieseniss S, Zahler S, Preissner K, Engelmann B. Intravascular tissue factor initiates coagulation via circulating microvesicles and platelets. FASEB J. 2003; 17: 476-8.
40. Natalie K. and Wolfgang K. Biomarkers of outcome from cardiovascular disease. Current Opinion in Critical Care 2006; 12: 412-419.
41. Naumann E, van Rees AB, Onning G, Oste R, Wydra M, Mensink RP. Beta-glucan incorporated into a fruit drink effectively lowers serum LDL-cholesterol concentrations. Am J Clin Nutr. 2006; 83: 601-5.
42. Nicolosi R, Bell SJ, Bistrian BR, Greenberg I, Forse RA, Blackburn GL. Plasma lipid changes after supplementation with beta-glucan fiber from yeast. Am J Clin Nutr. 1999; 70: 208-12.
43. Osborne JA, Lento PH, Siegfried MR, Stahl GL, Fusman B, Lefer AM. Cardiovascular effects of acute hypercholesterolemia in rabbits. Reversal with lovastatin treatment. J Clin Invest. 1989; 83: 465-73.
44. �敧terud B, Bj�廨klid E. Sources of tissue factor. Semin Thromb Hemost. 2006; 32: 11-23.
45. Panes O, Matus V, S�骸z CG, Quiroga T, Pereira J, Mezzano D. Human platelets synthesize and express functional tissue factor. Blood. 2007; 109: 5242-50.
46. Paul O., Jin Y., Sao-Tah F., and Nigel M. Retinoic acid selectively inhibits lipopolysaccharide induction of tissue factor gene expression in human monocytes. Blood 1998; 91: 2857-2865.
47. Rao L. V. M. and Usha R. P. Tissue factor-factor VIIa signaling Arterioscler. Thromb. Vasc. Biol. 2005; 25: 47-56.
48. Roberta B., Marina C., Carmen C., Caterina A., Michael D. E. and Elena T. Fluvastatin reduces tissue factor expression and macrophage accumulation in carotid lesions of cholesterol-fed rabbits in the absence of lipid lowering. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 692-698.
49. Roberta B., Marina C., Carmen C., Caterina A., Michael D. E. and Elena T., Fluvastatin reduces tissue factor expression and macrophage accumulation in carotid lesions of cholesterol-fed rabbits in the absence of lipid lowering. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 692-698.
50. Southgate DA. Digestion and metabolism of sugars. Am J Clin Nutr. 1995; 62: 203S-210S.
51. Spronk HM, van der Voort D, Ten Cate H. Blood coagulation and the risk of atherothrombosis: a complex relationship. Thromb J. 2004; 2: 12.
52. Stampfuss J. J., Censarek P., Fischer J. W., Schr�宁 K. and Weber A. A. Rapid release of active tissue factor from human arterial smooth muscle cells under flow conditions. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 34-e37.
53. Steffel J., L�卲cher T. F. and Tanner F. C. Tissue factor in cardiovascular diseases: molecular mechanisms and clinical implications. Circulation 2006; 113: 722-731
54. Stegnar M, Vene N, Bozic M. Do haemostasis activation markers that predict cardiovascular disease exist? Pathophysiol Haemost Thromb. 2003; 5: 302-8.
55. Steinemann S., Ulevitch RJ. and Mackman N., Role of the lipopolysaccharide (LPS) - binding protein / CD14 pathway in LPS induction of tissue factor expression in monocytic cells. Arterioscler. Thromb. Vasc. Biol. 1994; 14: 1202-1209.
56. Stevens RJ, Douglas KM, Saratzis AN, Kitas GD. Inflammation and atherosclerosis in rheumatoid arthritis. Expert Rev Mol Med. 2005; 7: 1-24.
57. Theuwissen E, Mensink RP. Simultaneous intake of beta-glucan and plant stanol esters affects lipid metabolism in slightly hypercholesterolemic subjects. J Nutr. 2007; 137: 583-8.
58. Theuwissen E, Mensink RP. Water-soluble dietary fibers and cardiovascular disease. Physiol Behav. 2008; 94: 285-92.
59. Tilman M. H., Ser�� K. M., Guido T., and Jan R. Protein S stimulates inhibition of the tissue factor pathway by tissue factor pathway inhibitor. PNAS 2006; 103: 3106-3111.
60. Topping DL, Clifton PM. Short-chain fatty acids and human colonic function: roles of resistant starch and nonstarch polysaccharides. Physiol Rev. 2001; 81: 1031-64.
61. Tsukagoshi S, Hashimoto Y, Fujii G, Kobayashi H, Nomoto K, Orita K. Krestin (PSK). Cancer Treat Rev. 1984; 11: 131-55.
62. Vicenta L. C. and Lina B., LDL Receptor-related protein and the vascular wall: implications for atherothrombosis. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 497-504.
63. Wilson TA, Nicolosi RJ, Delaney B, Chadwell K, Moolchandani V, Kotyla T, Ponduru S, Zheng GH, Hess R, Knutson N, Curry L, Kolberg L, Goulson M, Ostergren K. Reduced and high molecular weight barley beta-glucans decrease plasma total and non-HDL-cholesterol in hypercholesterolemic Syrian golden hamsters. J Nutr. 2004; 134: 2617.