跳到主要內容

臺灣博碩士論文加值系統

(3.95.131.146) 您好!臺灣時間:2021/07/25 14:53
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:陳松年
研究生(外文):Sung-Nien Chen
論文名稱:透過新合成n-3 eicosatrienoic acid對於經脂多醣體誘發小鼠巨噬細胞的抗發炎活性之影響
論文名稱(外文):Anti-inflammatory effects of newly synthesized n-3 eicosatrienoic acid in LPS-activated murine RAW264.7 macrophages
指導教授:莊路德
指導教授(外文):Lu-Te Chuang
學位類別:碩士
校院名稱:元培科技大學
系所名稱:生物技術研究所
學門:醫藥衛生學門
學類:其他醫藥衛生學類
論文種類:學術論文
畢業學年度:102
語文別:中文
論文頁數:105
中文關鍵詞:二十碳三烯酸n-3多元不飽和脂肪酸巨噬細胞發炎反應
外文關鍵詞:Eicosatrienoic acidn-3 polyunsaturated fatty acidMacrophageInflammation
相關次數:
  • 被引用被引用:1
  • 點閱點閱:185
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
二十碳三烯酸 (Eicosatrienoic acid; ETrA; Δ11,14,17-20:3) 為一種罕見的 n-3 多元不飽和脂肪酸 (n-3 polyunsatruated fatty acid; n-3 PUFA),是必需脂肪酸 α-次亞麻油酸 (Alpha-linolenic acid; ALA; Δ9,12,15-18:3)經加長作用而形成,由於在自然界分布不廣,鮮少被學術界所重視。先前研究指出: n-3 PUFA具有降低發炎反應的功效,因此,我們推測同為 n-3 PUFA 的二十碳三烯酸,也具有減緩發炎反應的功能。利用有機合成法將α-次亞麻油酸製造成二十碳三烯酸 (ETrA)再以管柱層析取得高純度ETrA,藉由Time-course與Dose-responce的實驗方法檢測二十碳三烯酸對小鼠巨噬細胞株RAW264.7細胞磷脂質脂肪酸組成的影響。以脂多醣體 (Lipopolysaccharide; LPS)刺激小鼠巨噬細胞RAW264.7,檢視並比較ETrA與其他n-3 多元不飽和脂肪酸能否抑制促發炎因子的生成與表現,包括:一氧化氮 (Nitric Oxide; NO)、前列腺素E2 (Prostaglandin; PGE2)、介白素-6 (Interlukin-6; IL-6)、腫瘤壞死因子 (Tumor Necrosis Factor-α; TNF-α)、一氧化氮合成酶 (In-ducible nitric oxide; iNOS)與第二型環氧合酶 (Cyclooxygenase type II; COX-2),再檢測細胞核內轉錄因子 (Nuclear transcription factor-kappa B; NF-κB)的活化是否被抑制。實驗結果顯示,ETrA在200 μM濃度內並不明顯影響細胞增殖,判定ETrA低於200 μM是不具有細胞毒殺作用。在細胞磷脂質組成中,隨著時間與劑量的增加ETrA與其代謝產物可取代n-6 PUFA中亞麻油酸 (LA)、花生四烯酸 (Arachidonic acid; AA)、Dihomo--linolenic acid (DGLA)與Docosapentaenoic acid (n-6 DPA)的比例,卻增加整體n-3 PUFA的含量。細胞發炎實驗中ETrA可透過降低iNOS表現而減少NO的生成,而PGE2、IL-6與TNF-α的生成並無顯著性的差異,並且不顯著影響胞內COX-2的表現量。此外,ETrA可透過調節核內轉錄因子 (NF-κB)的活化,減少p65次單元進入細胞核中。上述的實驗結果指出ETrA可取代細胞磷脂質脂肪酸的組成,減少n-6 PUFA的比例,並調節核內轉錄因子 (NF-κB)的訊號傳遞路徑而減少下游發炎因子iNOS與NO的表現與生成。實驗結果除了確認ETrA的抗發炎作用機制,也可運用於保健食品與醫療保健品的開發。
Eicosatrienoic acid (Δ11,14,17-20:3; ETrA), a unusual natural-ly-occurring n-3 polyunsaturated fatty acid (PUFA), is converted from the essential fatty acid α-linolenic acid (Δ9,12,15-18:3; ALA) by the action of elongase. Since n-3 PUFA has been reported to exert anti-inflammatory ef-fect, we speculated whether n-3 PUFA ETrA might also modulate immune responses. The objectives of this study were to synthesize and purify ETrA from alpha-linolenic acid (ALA) using combined techniques of chemical C2-elongation and column chromatography, and to determine whether ETrA modulated the n-6 PUFA composition in cellular phospholipids. Fur-thermore, the modulatory effect of ETrA and other n-3 PUFA on the pro-duction of pro-inflammatory mediators and signaling, such as nitric oxide (NO), prostaglandin E2 (PGE2), interlukin-6 (IL-6), tumor necrosis factor-α (TNF-α), inducible nitric oxide synthetase (iNOS), cyclooxygenase-2 (COX-2) and nuclear factor factor-kappa B (NF-κB) of murine macro-phages in response to lipopolysaccharide (LPS) was examined. Results showed that there was no cytotoxicity when the ETrA concentration was less than 200 μM. ETrA was taken up, incorporated and metabolized by macrophages, and the proportions of cellular phospholipid ETrA and its metabolites increased in a dose-dependent manner. The incorporation of ETrA also decreased the proportions of linoleic acid (LA), dihomo-γ-linolenic acid (DGLA) and arachidonic acid (AA) as well, and reduced the proportion of total n-6 PUFA and monounsaturated fatty acids (MUFA). ETrA suppressed the production of NO and expression of iNOS in LPS-stimulated macrophages, respectively. However, no significant de-crease in the production of PGE2, IL-6 and TNF-α was observed. The mod-ulation of NO synthesis and iNOS expression was due, in part, to the inactivation of nuclear factor-kappa B (NF-κB). Results of the present study provided a better understanding of the function mechanisms and modula-tory effect of ETrA on inflammation, and useful information for the future development of medical uses and nutritional supplementation.
誌謝 ................................................................................................................ I
中文摘要 ....................................................................................................... II
英文摘要 ..................................................................................................... IV
目錄 ............................................................................................................. VI
圖目錄 .......................................................................................................... X
表目錄 ........................................................................................................ XII
第一章 前言 ................................................................................................. 1
1.1 多元不飽和脂肪酸 (Polyunsatrated fatty acids; PUFAs).................... 1
1.1.1 ω-3與ω-6多元不飽和脂肪酸 ..................................................... 2
1.2 亞麻籽油 (Flexseed oil) ........................................................................ 6
1.2.1 α-次亞麻油酸 (Alpha-linolenic acid; ALA) ................................. 7
1.3 二十碳三烯酸 (Eicosatrienoic acid; ETrA) ......................................... 9
1.4 多元不飽和脂肪酸代謝路徑 (Polyunsatrated fatty acids metabolic pathway) ............................................................................... 11
1.5 發炎反應 (Inflammation).................................................................... 14
1.5.1 巨噬細胞 (Macrophages) .......................................................... 15
1.5.2 自由基 (Free radicals) ............................................................... 17
1.5.3 促發炎細胞激素 (Pro-inflammatory cytokines) ....................... 20
1.5.4 脂質衍生物 (Lipid derivatives) ................................................. 23
1.6 發炎路徑 (Inflammation pathway) ..................................................... 25
1.6.1 核轉錄因子 (Nuclear transcription factor kappa B; NF-κB) .... 26
1.7 實驗架構 .............................................................................................. 28
1.8 實驗目的 ............................................................................................ 29
第二章 材料與方法 ................................................................................... 30
2.1 儀器與設備 .......................................................................................... 30
2.2 材料 ...................................................................................................... 32
2.2.1 油脂&脂肪酸 .............................................................................. 32
2.2.2 藥品 ............................................................................................. 32
2.2.3 免疫試劑套組 (Enzyme-linked immunosorbent assay kit) ...... 35
2.3 實驗細胞株 .......................................................................................... 36
2.3.1 細胞培養與繼代 ......................................................................... 36
2.3.2 細胞株活化 ................................................................................. 37
2.3.3 細胞株保存 ................................................................................. 37
2.3.4 細胞株計數 ................................................................................. 38
2.4 二十碳三烯酸 (ETrA)脂肪酸製備 .................................................... 39
2.4.1 長碳鏈脂肪酸化學合成步驟
..................................................... 39
2.4.2 脂肪酸甲基酯化 ......................................................................... 44
2.4.3 脂肪酸皂化 ................................................................................. 45
2.4.4 薄層層析法 ................................................................................. 46
2.4.5 管柱層析法 ................................................................................. 46
2.4.6 銀離子管柱層析法 ..................................................................... 47
2.4.7 脂質萃取 ..................................................................................... 47
2.4.8 氣相層析儀-脂肪酸分析 ........................................................... 48
2.5脂肪酸配製 ........................................................................................... 49
2.5.1細胞實驗-脂肪酸濃度配製 ........................................................ 49
2.6 以巨噬細胞株探討新合成的ω-3多元不飽和脂肪酸 (ETrA) ........ 50
2.6.1 細胞存活率 ................................................................................. 50
2.6.2 脂肪酸代謝 (Time) .................................................................... 51
2.6.3 脂肪酸代謝 (Dose) .................................................................... 52
2.7 以脂多醣體誘導巨噬細胞株探討ETrA脂肪酸對發炎指標的影響 ....................................................................................................... 53
2.7.1 一氧化碳釋放量 ......................................................................... 53
2.7.2 介白素-6 ..................................................................................... 54
2.7.3 腫瘤壞死因子-α測定 ................................................................ 56
2.7.4 前列腺素E2測定 ....................................................................... 57
2.7.5 蛋白質萃取與定量 ..................................................................... 59
2.7.6 SDS-PAGE/Western Blot ............................................................. 60
2.8 以脂多醣體誘導巨噬細胞探討ETrA脂肪酸對細胞核內發炎路徑的影響 ......................................................................................... 62
2.8.1 核內轉錄因子-κB活性測定...................................................... 62
2.8 統計分析 .............................................................................................. 65
第三章 結果與討論 ................................................................................... 63
3.1 二十碳三烯酸 (ETrA)合成 ................................................................ 66
3.2 新合成脂ETrA對於細胞存活率的影響 ........................................... 73
3.3 ETrA對於細胞磷脂質中脂肪酸組成與代謝的影響 ........................ 75
3.4 探討ETrA之抗發炎作用與機制 ....................................................... 81
3.4.1 ETrA抑制經LPS誘導巨噬細胞之一氧化氮 (NO)生成量 .... 81
3.4.2 ETrA抑制經LPS誘導巨噬細胞之前列腺素E2 (PGE2)生成量 ....................................................................................................... 82
3.4.3 ETrA抑制經LPS誘導之腫瘤壞死因子 (TNF-α)的生成 ....... 84
3.4.4 ETrA抑制經LPS誘導之介白素-6 (IL-6)的生成 .................... 85
3.4.5 ETrA抑制巨噬細胞之一氧化氮合成酶 (iNOS)蛋白質的表現 ................................................................................................... 86
3.4.6 ETrA抑制巨噬細胞之第二型環氧合酶 (COX-2)蛋白質的表現 ................................................................................................... 87
3.4.7 ETrA抑制細胞內發炎路徑之核轉錄因子 (NF-κB)的活化.... 88
第四章 結論 ............................................................................................... 91
參考文獻 ..................................................................................................... 94
附錄 ........................................................................................................... 100
1. Adkins, Y., and D.S. Kelley. 2010. Mechanisms underlying the cardioprotective effects of omega-3 polyunsaturated fatty acids. The Journal of nutritional biochemistry. 21:781-792.
2. Arana, L., P. Gangoiti, A. Ouro, I.G. Rivera, M. Ordonez, M. Trueba, R.S. Lankalapalli, R. Bittman, and A. Gomez-Munoz. 2012. Generation of reactive oxygen species (ROS) is a key factor for stimulation of macrophage proliferation by ceramide 1-phosphate. Exp Cell Res. 318:350-360.
3. Balistreri, C.R., C. Caruso, F. Listi, G. Colonna-Romano, D. Lio, and G. Candore. 2011. LPS-mediated production of pro/anti-inflammatory cytokines and eicosanoids in whole blood samples: biological effects of +896A/G TLR4 polymorphism in a Sicilian population of healthy subjects. Mechanisms of ageing and development. 132:86-92.
4. Barcelo-Coblijn, G., and E.J. Murphy. 2009. Alpha-linolenic acid and its conversion to longer chain n-3 fatty acids: benefits for human health and a role in maintaining tissue n-3 fatty acid levels. Progress in lipid research. 48:355-374.
5. Calder, P.C. 1997. N-3 polyunsaturated fatty acids and immune cell function. Advances in enzyme regulation. 37:197-237.
6. Calder, P.C. 2009. Polyunsaturated fatty acids and inflammatory processes: New twists in an old tale. Biochimie. 91:791-795.
7. Caughey, G.E., E. Mantzioris, R.A. Gibson, L.G. Cleland, and M.J. James. 1996. The effect on human tumor necrosis factor alpha and interleukin 1 beta production of diets enriched in n-3 fatty acids from vegetable oil or fish oil. The American journal of clinical nutrition. 63:116-122.
8. Chen, E.P., and E.M. Smyth. 2011. COX-2 and PGE2-dependent immunomodulation in breast cancer. Prostaglandins & other lipid mediators. 96:14-20.
9. Cianciulli, A., R. Calvello, P. Cavallo, T. Dragone, V. Carofiglio, and M.A. Panaro. 2012. Modulation of NF-kappaB activation by resveratrol in LPS treated human intestinal cells results in downregulation of PGE2 production and COX-2 expression. Toxicology in vitro : an international journal published in association with BIBRA. 26:1122-1128.
10. Cunnane, S.C., and M.J. Anderson. 1997. Pure linoleate deficiency in the rat: influence on growth, accumulation of n-6 polyunsaturates, and [1-14C]linoleate oxidation. Journal of lipid research. 38:805-812.
11. Denlinger, L.C., P.L. Fisette, K.A. Garis, G. Kwon, A. Vazquez-Torres, A.D. Simon, B. Nguyen, R.A. Proctor, P.J. Bertics, and J.A. Corbett. 1996. Regulation of inducible nitric oxide synthase expression by macrophage purinoreceptors and calcium. The Journal of biological chemistry. 271:337-342.
12. Dhopeshwarkar, G.A., C. Subramanian, and J.F. Mead. 1971. RAPID UPTAKE OF l-$sup 14$C ACETATE BY THE ADULT RAT BRAIN 15 SECONDS AFTER CAROTID INJECTION. In Other Information: UNCL. Orig. Receipt Date: 31-DEC-71. Medium: X.
13. Diggle, C.P. 2002. In vitro studies on the relationship between polyunsaturated fatty acids and cancer: tumour or tissue specific effects? Progress in lipid research. 41:240-253.
14. Fawzy, A.A., B.S. Vishwanath, and R.C. Franson. 1988. Inhibition of human non-pancreatic phospholipases A2 by retinoids and flavonoids. Mechanism of action. Agents and actions. 25:394-400.
15. Horiuchi, T., H. Mitoma, S. Harashima, H. Tsukamoto, and T. Shimoda. 2010. Transmembrane TNF-alpha: structure, function and interaction with anti-TNF agents. Rheumatology (Oxford). 49:1215-1228.
16. Huang, A., C. Li, R.L. Kao, and W.L. Stone. 1999. Lipid hydroperoxides inhibit nitric oxide production in RAW264.7 macrophages. Free radical biology & medicine. 26:526-537.
17. Huang, Y.S., W.C. Huang, C.W. Li, and L.T. Chuang. 2011. Eicosadienoic acid differentially modulates production of pro-inflammatory modulators in murine macrophages. Mol Cell Biochem. 358:85-94.
18. Jin, X.J., E.J. Kim, I.K. Oh, Y.K. Kim, C.H. Park, and J.H. Chung. 2010. Prevention of UV-induced skin damages by 11,14,17-eicosatrienoic acid in hairless mice in vivo. Journal of Korean medical science. 25:930-937.
19. Kalinski, P. 2012. Regulation of immune responses by prostaglandin E2. J Immunol. 188:21-28.
20. Kim, B.H., K.S. Kang, and Y.S. Lee. 2004. Effect of retinoids on LPS-induced COX-2 expression and COX-2 associated PGE(2) release from mouse peritoneal macrophages and TNF-alpha release from rat peripheral blood mononuclear cells. Toxicology letters. 150:191-201.
21. Kim, B.H., K.H. Lee, E.Y. Chung, Y.S. Chang, H. Lee, C.K. Lee, K.R. Min, and Y. Kim. 2006. Inhibitory effect of chroman carboxamide on interleukin-6 expression in response to lipopolysaccharide by preventing nuclear factor-kappaB activation in macrophages. European journal of pharmacology. 543:158-165.
22. Kim, E.J., M.K. Kim, X.J. Jin, J.H. Oh, J.E. Kim, and J.H. Chung. 2010. Skin aging and photoaging alter fatty acids composition, including 11,14,17-eicosatrienoic acid, in the epidermis of human skin. Journal of Korean medical science. 25:980-983.
23. Langelier, B., J.M. Alessandri, M.H. Perruchot, P. Guesnet, and M. Lavialle. 2005. Changes of the transcriptional and fatty acid profiles in response to n-3 fatty acids in SH-SY5Y neuroblastoma cells. Lipids. 40:719-728.
24. Martin, C.A., and M.E. Dorf. 1991. Differential regulation of interleukin-6, macrophage inflammatory protein-1, and JE/MCP-1 cytokine expression in macrophage cell lines. Cellular immunology. 135:245-258.
25. Mustonen, H., A. Hietaranta, P. Puolakkainen, E. Kemppainen, H. Paimela, T. Kiviluoto, and E. Kivilaakso. 2007. Ethanol induced NF-{kappa}B activation protects against cell injury in cultured rat gastric mucosal epithelium. American journal of physiology. Gastrointestinal and liver physiology. 292:G1614-1621.
26. Novak, T.E., T.A. Babcock, D.H. Jho, W.S. Helton, and N.J. Espat. 2003. NF-kappa B inhibition by omega -3 fatty acids modulates LPS-stimulated macrophage TNF-alpha transcription. American journal of physiology. Lung cellular and molecular physiology. 284:L84-89.
27. Opal, S.M., and V.A. DePalo. 2000. Anti-inflammatory cytokines. Chest. 117:1162-1172.
28. Park, J.Y., M.H. Pillinger, and S.B. Abramson. 2006. Prostaglandin E2 synthesis and secretion: the role of PGE2 synthases. Clin Immunol. 119:229-240.8
29. Peri, F., and M. Piazza. 2012. Therapeutic targeting of innate immunity with Toll-like receptor 4 (TLR4) antagonists.
Biotechnology advances. 30:251-260.
30. Peters, M., S. Jacobs, M. Ehlers, P. Vollmer, J. Mullberg, E. Wolf, G. Brem, K.H. Meyer zum Buschenfelde, and S. Rose-John. 1996. The function of the soluble interleukin 6 (IL-6) receptor in vivo: sensitization of human soluble IL-6 receptor transgenic mice towards IL-6 and prolongation of the plasma half-life of IL-6. The Journal of experimental medicine. 183:1399-1406.
31. Petersen, A.M., and B.K. Pedersen. 2005. The anti-inflammatory effect of exercise. J Appl Physiol. 98:1154-1162.
32. Portolesi, R., B.C. Powell, and R.A. Gibson. 2008. Delta6 desaturase mRNA abundance in HepG2 cells is suppressed by unsaturated fatty acids. Lipids. 43:91-95.
33. Saitoh, S. 2009. Chaperones and transport proteins regulate TLR4 trafficking and activation. Immunobiology. 214:594-600.
34. Schwab, U.S., J.C. Callaway, A.T. Erkkila, J. Gynther, M.I. Uusitupa, and T. Jarvinen. 2006. Effects of hempseed and flaxseed oils on the profile of serum lipids, serum total and lipoprotein lipid concentrations and haemostatic factors. European journal of nutrition. 45:470-477.
35. Serhan, C.N., C.B. Clish, J. Brannon, S.P. Colgan, K. Gronert, and N. Chiang. 2000. Anti-microinflammatory lipid signals generated from dietary N-3 fatty acids via cyclooxygenase-2 and transcellular processing: a novel mechanism for NSAID and N-3 PUFA therapeutic actions. Journal of physiology and pharmacology : an official journal of the Polish Physiological Society. 51:643-654.
36. Shen, S.C., W.R. Lee, H.Y. Lin, H.C. Huang, C.H. Ko, L.L. Yang, and Y.C. Chen. 2002. In vitro and in vivo inhibitory activities of rutin, wogonin, and quercetin on lipopolysaccharide-induced nitric oxide and prostaglandin E(2) production. European journal of pharmacology. 446:187-194.
37. Simpson, R.J., A. Hammacher, D.K. Smith, J.M. Matthews, and L.D. Ward. 1997. Interleukin-6: structure-function relationships. Protein science : a publication of the Protein Society. 6:929-955.
38. Steffen, Y., T. Jung, L.O. Klotz, T. Schewe, T. Grune, and H. Sies. 2007. Protein modification elicited by oxidized low-density lipoprotein (LDL) in endothelial cells: protection by (-)-epicatechin. Free radical biology & medicine. 42:955-970.
39. Van Hauwermeiren, F., R.E. Vandenbroucke, and C. Libert. 2011.Treatment of TNF mediated diseases by selective inhibition of soluble TNF or TNFR1. Cytokine & growth factor reviews. 22:311-319.
40. Westermarck, J., S.P. Li, T. Kallunki, J. Han, and V.M. Kahari. 2001. p38 mitogen-activated protein kinase-dependent activation of protein phosphatases 1 and 2A inhibits MEK1 and MEK2 activity and collagenase 1 (MMP-1) gene expression. Molecular and cellular biology. 21:2373-2383.
41. Wilkinson, P., C. Leach, E.E. Ah-Sing, N. Hussain, G.J. Miller, D.J. Millward, and B.A. Griffin. 2005. Influence of alpha-linolenic acid and fish-oil on markers of cardiovascular risk in subjects with an atherogenic lipoprotein phenotype. Atherosclerosis. 181:115-124.
42. Yun, K.J., J.S. Shin, J.H. Choi, N.I. Back, H.G. Chung, and K.T. Lee. 2009. Quaternary alkaloid, pseudocoptisine isolated from tubers of Corydalis turtschaninovi inhibits LPS-induced nitric oxide, PGE(2), and pro-inflammatory cytokines production via the down-regulation of NF-kappaB in RAW 264.7 murine macrophage cells. International immunopharmacology. 9:1323-1331.
43. Zhang, Y., H. Chen, and L. Yang. 2010. Toll-like receptor 4 participates in gastric mucosal protection through Cox-2 and PGE2. Digestive and liver disease : official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver. 42:472-476.
44. Zhao, G., T.D. Etherton, K.R. Martin, J.P. Vanden Heuvel, P.J. Gillies, S.G. West, and P.M. Kris-Etherton. 2005. Anti-inflammatory effects of polyunsaturated fatty acids in THP-1 cells. Biochemical and biophysical research communications. 336:909-917.
45. Zhao, Y., S. Joshi-Barve, S. Barve, and L.H. Chen. 2004. Eicosapentaenoic acid prevents LPS-induced TNF-alpha expression by preventing NF-kappaB activation. Journal of the American College of Nutrition. 23:71-78.
46. 謝明哲、胡淼琳、楊素卿、陳俊榮、徐成金、陳明汝,食用營養學 Practical Nutrition,華杏出版股份有限公司,民國97年
47. 徐成金、吳志忠、盧義發、蕭慧美、魏明敏、張菡馨、黃慧煐、
楊新玲、吳淑惠、林錦華、吳孟純、陳宣潁、吳素珍、趙碧玉,營養學 (三版) Nutrition,華格那企業股份有限公司,民國101年
48. 鄧富元、龔素芳、夏毅然、高文斌、謝耀東,「NF-κB在發炎反應、細胞凋亡與癌症生成過程中的調控」,Chin Dent J (中華牙誌) 25(1):12-24 March 2006
49. 蕭寧馨 編譯,透視營養學,美商麥格羅˙希爾國際股份有限公司 台灣分公司、藝軒圖書出版社,民國100年
連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊