近年來許多研究指出，腸道菌叢不平衡及腸道通透性增加為酒精性
肝臟疾病(alcoholic liver disease)之重要致病機轉之一。而魚油在許多研究中也被發現具有抗發炎及抗氧化等功效。因此本研究以腸道健康的觀點，探討魚油對於大白鼠酒精性肝臟疾病之改善效果。本研究使用八週齡雄性Wistar 大白鼠48 隻，經一週預養後，依據血漿AST(Aspartateaminotransferase)、ALT(Alanineaminotransferase)活性分為控制組(C)、以魚油取代25%橄欖油組(CF25)、魚油取代57%橄欖油組(CF57)、酒精組(E)、以魚油取代25%橄欖油酒精組(EF25)、魚油取代57%橄欖油酒精組(EF57)等六組。控制組以不含酒精之液體飼料餵養，酒精組以酒精液體飼料餵養，而CF25、EF25 組以及CF57、EF57 組則分別取代控制組或酒精組飼料中25%及57%的橄欖油，各組以等熱量方式進行餵養。於實驗期第八週進行腸道通透性測定及收集糞便樣本進行菌相分析。實驗結果發現，肝功能指標AST 及ALT 活性在第八週時E 組皆顯著高於C組，而與Ｅ組相比，EF25 及EF57 組其AST 活性皆顯著下降。肝臟病理切片結果E 組脂肪堆積、發炎以及壞死程度方面皆顯著高於C 組；與Ｅ組比較，EF25 及EF57 則可以顯著降低脂肪堆積及發炎反應。氧化壓力測
定方面，肝臟中GSH/GSSG ratio 方面，C 組與E 組無顯著差異，與Ｅ
組相比EF25 組無顯著差異，但EF57 組則顯著上升。血漿及肝臟TBARS濃度，E 組皆顯著高於C 組，而與Ｅ組相比，EF25 及EF57 組皆顯著降低。肝臟CYP2E1 表現量，E 組表現量顯著高於C 組，Ｅ組與EF25 及EF57 組相比則無顯著差異。發炎反應方面，E 組肝臟中TNF-α、IL-1、IL-6、IL-10 細胞激素濃度皆顯著高於C 組，與Ｅ組相比EF25 及EF57組肝臟IL-1、IL-6、IL-10 濃度顯著降低，然而EF25 組其TNF-α 濃度顯著低於Ｅ組。血漿中內毒素含量各組間皆無顯著差異。腸道通透性各組間皆無顯著差異。腸道菌相方面，總厭氧菌及Lactobacilli 菌數各組間皆無顯著差異。E. coli 菌數方面，C 組與E 組雖無顯著差異，但與Ｅ組相比，EF25 及EF57 組皆顯著降低。此外，Bifidobacteria 菌數方面，Ｅ組顯著低於Ｃ組，與Ｅ組相比，EF25 組菌數顯著升高，但EF57 組則無顯著差異。由以上實驗結果可知，以25%或57%魚油取代飼料中橄欖油，可藉由降低長期攝取酒精之大白鼠肝臟TNF-α、IL-1、IL-6 以及IL-10濃度及降低腸道有害菌E.coli 並增加有益菌Bifidobacteria 菌數，以改善大白鼠酒精性肝損傷。

Imbalance of intestinal microbiota and increased intestinal permeability are considered as important pathological development of alcoholic liver
disease. The previous study was to investigate the fish oil have potential in anti-inflammatory and anti-oxidative. Therefore, the present study was to
investigate the protective effects of fish oil on hepatic injury in ethanol-fed rats based on intestinal health. The 8-weeks-old male Wistar rats were acclimated for 1 week, and then according to aspartate aminotransferase
(AST) and alanine aminotransferase (ALT) activities, 48 rats divided into six
groups (n=8 per group) for feeding with either control diet or ethanol diet, in
which the fat composition of both diets was adjusted with 25% or 57% fish oil substitution for olive oil. The groups were C (control), CF25 (control
with 25% fish oil substitution for olive oil), CF57 (control with 57% fish oil substitution for olive oil), E (ethanol), EF25 (ethanol with 25% fish oil substitution for olive oil), and EF57 (ethanol with 57% fish oil substitution for olive oil). Before sacrificed, will analysis the intestinal permeability and
intestinal microbiota. Rats were sacrificed after 8 weeks. Results showed that plasma AST and ALT activities, hepatic thiobarbituric acid reactive substances (TBARS) levels, and hepatic tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-10 were significantly increased in E group.
In addition, hepatic histopathological analysis scores of inflammation, degeneration and necrosis, and fatty change in E group were significantly
higher. However, plasma AST activity, plasma and hepatic TBARS, and hepatic TNF-α, IL-1β, IL-6, and IL-10 levels were significantly decreased in EF25 and EF57 groups than those in E group. The histological changes were
also improved in EF25 and EF57 groups. The fecal Lactobacilli numbers in E group were significantly decreased than that in C group. However, the
fecal Lactobacilli numbers in EF25 group were significantly increased than that in E group. There was no change in intestinal permeability among
groups. In conclusion, suggested that fish oil substitution may prevent ethanol-induced liver damage by reducing inflammatory cytokines and improving the intestinal microbiota composition.

目錄
中文摘要 I
英文摘要 III
致謝 IV
圖目次 VIII
表目錄 IX
第一章 前言與研究動機1
第二章 文獻回顧3
第一節 酒精代謝3
第二節 酒精性肝臟疾病致病機轉6
第三節 酒精攝取與腸道健康之關係13
第四節 n-3 多元不飽和脂肪酸的生理功能17
第五節 酒精性肝臟疾病與 n-3多元不飽和脂肪酸之相關研究20
第三章 實驗設計與方法21
第一節 實驗動物及分組21
第二節 抽血及犧牲 23
第三節 分析項目及方法26
第四節 統計方法 45
第四章 結果 46
第一節 攝食量及體重46
第二節 相對肝重 47
第三節 肝功能指標 49
第四節 氧化壓力 55
第五節 發炎反應 59
第六節 腸道健康 62
第五章、討論 65
第一節 攝食量及體重 65
第二節 相對肝重率 67
第三節 肝功能指標 68
第四節 氧化壓力 70
第五節 發炎反應 73
第六節 腸道健康 77
第七節 綜合討論 80
第六章、結論 83
參考文獻 84

圖目次
圖一、酒精在細胞中之代謝途徑5
圖二、內毒素結構11
圖三、內毒素、腸道通透性以及酒精性肝損傷之關係12
圖四、腸道緊密連結之組成16
圖五、實驗架構24
圖六、各組大鼠肝臟組織病理切片(Hematoxylin and eosin染色)53
圖七、各組大鼠肝臟組織病理切片(Masson trichrome染色)54
圖八、酒精餵食八週後各組大鼠肝臟CYP2E1蛋白表現量之影響56

 
表目錄
表一、實驗飼料組成25
表二、肝臟組織病理切片評分 28
表三 、魚油對於餵食酒精八週後大鼠體重、肝重以及相對肝重率影響 48
表四、魚油對於餵食酒精八週後大鼠血漿中AST及ALT活性之影響50
表五、各組大鼠肝臟病理切片評分52
表六、魚油對於餵食酒精八週後各組大鼠紅血球及肝臟GSH/GSSG 比值之影響57
表七、魚油對於餵食酒精八週後各組大鼠血漿及肝臟中TBARS含量之影響58
表八、魚油對於餵食酒精八週後各組大鼠肝臟中TNF-α、 IL-1β、IL-6 以及IL-10之影響60
表九、魚油對於餵食酒精八週後各組大鼠血漿內毒素之影響 61
表十、魚油對於餵食酒精八週後各組大鼠尿液中L/M比值影響63
表十一、魚油對於餵食酒精八週後各組大鼠糞便菌相之影響 64

1.Aliche-Djoudi, F., Podechard, N., Collin, A., Chevanne, M., Provost, E., Poul, M., Le Hegarat, L., Catheline, D., Legrand, P., Dimanche-Boitrel, M.T., et al. (2013). A role for lipid rafts in the protection afforded by docosahexaenoic acid against ethanol toxicity in primary rat hepatocytes. Food Chem Toxicol 60, 286-296.
2.Arrieta, M.C., Bistritz, L., and Meddings, J.B. (2006). Alterations in intestinal permeability. Gut 55, 1512-1520.
3.Bode, C., and Bode, J.C. (2005). Activation of the innate immune system and alcoholic liver disease: effects of ethanol per se or enhanced intestinal translocation of bacterial toxins induced by ethanol? Alcoholism, clinical and experimental research 29, 166s-171s.
4.Bode, J.C., Bode, C., Heidelbach, R., Durr, H.K., and Martini, G.A. (1984). Jejunal microflora in patients with chronic alcohol abuse. Hepatogastroenterology 31, 30-34.
5.Bull-Otterson, L., Feng, W., Kirpich, I., Wang, Y., Qin, X., Liu, Y., Gobejishvili, L., Joshi-Barve, S., Ayvaz, T., Petrosino, J., et al. (2013). Metagenomic analyses of alcohol induced pathogenic alterations in the intestinal microbiome and the effect of Lactobacillus rhamnosus GG treatment. PloS one 8, e53028.
6.Calder, P.C. (2008). The relationship between the fatty acid composition of immune cells and their function. Prostaglandins Leukot Essent Fatty Acids 79, 101-108.
7.Camandola, S., Leonarduzzi, G., Musso, T., Varesio, L., Carini, R., Scavazza, A., Chiarpotto, E., Baeuerle, P.A., and Poli, G. (1996). Nuclear factor kB is activated by arachidonic acid but not by eicosapentaenoic acid. Biochemical and biophysical research communications 229, 643-647.
8.Chen, P., and Schnabl, B. (2014). Host-Microbiome Interactions in Alcoholic Liver Disease. Gut Liver 8, 237-241.
9.Chen, Y.L., Peng, H.C., Hsieh, Y.C., and Yang, S.C. (2014). Epidermal growth factor improved alcohol-induced inflammation in rats. Alcohol 48, 701-706.
10.Chen, Y.L., Yang, S.S., Peng, H.C., Hsieh, Y.C., Chen, J.R., and Yang, S.C. (2011). Folate and vitamin B12 improved alcohol-induced hyperhomocysteinemia in rats. Nutrition 27, 1034-1039.
11.Chiu, W.C., Huang, Y.L., Chen, Y.L., Peng, H.C., Liao, W.H., Chuang, H.L., Chen, J.R., and Yang, S.C. (2015). Synbiotics reduce ethanol-induced hepatic steatosis and inflammation by improving intestinal permeability and microbiota in rats. Food Funct.
12.Cleland, L.G., James, M.J., and Proudman, S.M. (2006). Fish oil: what the prescriber needs to know. Arthritis research & therapy 8, 202.
13.Comporti, M., Signorini, C., Leoncini, S., Gardi, C., Ciccoli, L., Giardini, A., Vecchio, D., and Arezzini, B. (2010). Ethanol-induced oxidative stress: basic knowledge. Genes & nutrition 5, 101-109.
14.D''Souza El-Guindy, N.B., Kovacs, E.J., De Witte, P., Spies, C., Littleton, J.M., de Villiers, W.J., Lott, A.J., Plackett, T.P., Lanzke, N., and Meadows, G.G. (2010). Laboratory models available to study alcohol-induced organ damage and immune variations: choosing the appropriate model. Alcoholism, clinical and experimental research 34, 1489-1511.
15.Das, S.K., and Vasudevan, D.M. (2007). Alcohol-induced oxidative stress. Life Sci 81, 177-187.
16.Deaciuc, I.V., D''Souza, N.B., and Spitzer, J.J. (1995). Tumor necrosis factor-alpha cell-surface receptors of liver parenchymal and nonparenchymal cells during acute and chronic alcohol administration to rats. Alcoholism, clinical and experimental research 19, 332-338.
17.Decker, K. (1990). Biologically active products of stimulated liver macrophages (Kupffer cells). Eur J Biochem 192, 245-261.
18.Donnet-Hughes, A., Schiffrin, E.J., and Turini, M.E. (2001). The intestinal mucosa as a target for dietary polyunsaturated fatty acids. Lipids 36, 1043-1052.
19.Forsyth, C.B., Tang, Y., Shaikh, M., Zhang, L., and Keshavarzian, A. (2011). Role of snail activation in alcohol-induced iNOS-mediated disruption of intestinal epithelial cell permeability. Alcoholism, clinical and experimental research 35, 1635-1643.
20.French, S.W. (1993). Nutrition in the pathogenesis of alcoholic liver disease. Alcohol Alcohol 28, 97-109.
21.Gao, B. (2012). Hepatoprotective and anti-inflammatory cytokines in alcoholic liver disease. J Gastroenterol Hepatol 27 Suppl 2, 89-93.
22.Gao, B., and Bataller, R. (2011). Alcoholic liver disease: pathogenesis and new therapeutic targets. Gastroenterology 141, 1572-1585.
23.Gao, B., Seki, E., Brenner, D.A., Friedman, S., Cohen, J.I., Nagy, L., Szabo, G., and Zakhari, S. (2011). Innate immunity in alcoholic liver disease. American journal of physiology Gastrointestinal and liver physiology 300, G516-525.
24.Hartsock, A., and Nelson, W.J. (2008). Adherens and tight junctions: structure, function and connections to the actin cytoskeleton. Biochimica et biophysica acta 1778, 660-669.
25.Hezode, C., Lonjon, I., Roudot-Thoraval, F., Pawlotsky, J.M., Zafrani, E.S., and Dhumeaux, D. (2003). Impact of moderate alcohol consumption on histological activity and fibrosis in patients with chronic hepatitis C, and specific influence of steatosis: a prospective study. Aliment Pharmacol Ther 17, 1031-1037.
26.Hill, D.B., D''Souza, N.B., Lee, E.Y., Burikhanov, R., Deaciuc, I.V., and de Villiers, W.J. (2002). A role for interleukin-10 in alcohol-induced liver sensitization to bacterial lipopolysaccharide. Alcoholism, clinical and experimental research 26, 74-82.
27.Hoek, J.B., and Pastorino, J.G. (2002). Ethanol, oxidative stress, and cytokine-induced liver cell injury. Alcohol 27, 63-68.
28.Holzapfel, W.H., Haberer, P., Snel, J., Schillinger, U., and Huis in''t Veld, J.H. (1998). Overview of gut flora and probiotics. Int J Food Microbiol 41, 85-101.
29.Huang, C.C., Chen, J.R., Liu, C.C., Chen, K.T., Shieh, M.J., and Yang, S.C. (2005). Effects of long-term ethanol consumption on jejunal lipase and disaccharidase activities in male and female rats. World journal of gastroenterology : WJG 11, 2603-2608.
30.Ishikado, A., Morino, K., Nishio, Y., Nakagawa, F., Mukose, A., Sono, Y., Yoshioka, N., Kondo, K., Sekine, O., Yoshizaki, T., et al. (2013). 4-Hydroxy hexenal derived from docosahexaenoic acid protects endothelial cells via Nrf2 activation. PloS one 8, e69415.
31.Kawai, T., and Akira, S. (2007). TLR signaling. Seminars in immunology 19, 24-32.
32.Kawase, T., Kato, S., and Lieber, C.S. (1989). Lipid peroxidation and antioxidant defense systems in rat liver after chronic ethanol feeding. Hepatology 10, 815-821.
33.Keshavarzian, A., Choudhary, S., Holmes, E.W., Yong, S., Banan, A., Jakate, S., and Fields, J.Z. (2001). Preventing gut leakiness by oats supplementation ameliorates alcohol-induced liver damage in rats. J Pharmacol Exp Ther 299, 442-448.
34.Keshavarzian, A., Farhadi, A., Forsyth, C.B., Rangan, J., Jakate, S., Shaikh, M., Banan, A., and Fields, J.Z. (2009). Evidence that chronic alcohol exposure promotes intestinal oxidative stress, intestinal hyperpermeability and endotoxemia prior to development of alcoholic steatohepatitis in rats. J Hepatol 50, 538-547.
35.Keshavarzian, A., Holmes, E.W., Patel, M., Iber, F., Fields, J.Z., and Pethkar, S. (1999). Leaky gut in alcoholic cirrhosis: a possible mechanism for alcohol-induced liver damage. Am J Gastroenterol 94, 200-207.
36.Kikugawa, K., Yasuhara, Y., Ando, K., Koyama, K., Hiramoto, K., and Suzuki, M. (2003). Protective effect of supplementation of fish oil with high n-3 polyunsaturated fatty acids against oxidative stress-induced DNA damage of rat liver in vivo. J Agric Food Chem 51, 6073-6079.
37.Kirpich, I.A., Solovieva, N.V., Leikhter, S.N., Shidakova, N.A., Lebedeva, O.V., Sidorov, P.I., Bazhukova, T.A., Soloviev, A.G., Barve, S.S., McClain, C.J., et al. (2008). Probiotics restore bowel flora and improve liver enzymes in human alcohol-induced liver injury: a pilot study. Alcohol 42, 675-682.
38.Kleiner, D.E., Brunt, E.M., Van Natta, M., Behling, C., Contos, M.J., Cummings, O.W., Ferrell, L.D., Liu, Y.C., Torbenson, M.S., Unalp-Arida, A., et al. (2005). Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 41, 1313-1321.
39.Li, H., Ruan, X.Z., Powis, S.H., Fernando, R., Mon, W.Y., Wheeler, D.C., Moorhead, J.F., and Varghese, Z. (2005). EPA and DHA reduce LPS-induced inflammation responses in HK-2 cells: evidence for a PPAR-gamma-dependent mechanism. Kidney international 67, 867-874.
40.Lieber, C.S. (2004). Alcoholic fatty liver: its pathogenesis and mechanism of progression to inflammation and fibrosis. Alcohol 34, 9-19.
41.Lu, Y., and Cederbaum, A.I. (2006). Enhancement by pyrazole of lipopolysaccharide-induced liver injury in mice: role of cytochrome P450 2E1 and 2A5. Hepatology 44, 263-274.
42.Malaguarnera, G., Giordano, M., Nunnari, G., Bertino, G., and Malaguarnera, M. (2014). Gut microbiota in alcoholic liver disease: pathogenetic role and therapeutic perspectives. World journal of gastroenterology : WJG 20, 16639-16648.
43.Marshall, J.C., Walker, P.M., Foster, D.M., Harris, D., Ribeiro, M., Paice, J., Romaschin, A.D., and Derzko, A.N. (2002). Measurement of endotoxin activity in critically ill patients using whole blood neutrophil dependent chemiluminescence. Critical care (London, England) 6, 342-348.
44.McClain, C.J., Barve, S., Deaciuc, I., Kugelmas, M., and Hill, D. (1999). Cytokines in alcoholic liver disease. Semin Liver Dis 19, 205-219.
45.Mezey, E., Caballeria, J., Mitchell, M.C., Pares, A., Herlong, H.F., and Rodes, J. (1991). Effect of parenteral amino acid supplementation on short-term and long-term outcomes in severe alcoholic hepatitis: a randomized controlled trial. Hepatology 14, 1090-1096.
46.Nanji, A.A. (2004). Role of different dietary fatty acids in the pathogenesis of experimental alcoholic liver disease. Alcohol 34, 21-25.
47.Niessen, C.M. (2007). Tight junctions/adherens junctions: basic structure and function. The Journal of investigative dermatology 127, 2525-2532.
48.Parlesak, A., Schafer, C., Schutz, T., Bode, J.C., and Bode, C. (2000). Increased intestinal permeability to macromolecules and endotoxemia in patients with chronic alcohol abuse in different stages of alcohol-induced liver disease. J Hepatol 32, 742-747.
49.Patten, A.R., Brocardo, P.S., and Christie, B.R. (2013). Omega-3 supplementation can restore glutathione levels and prevent oxidative damage caused by prenatal ethanol exposure. J Nutr Biochem 24, 760-769.
50.Petrasek, J., Dolganiuc, A., Csak, T., Nath, B., Hritz, I., Kodys, K., Catalano, D., Kurt-Jones, E., Mandrekar, P., and Szabo, G. (2011). Interferon regulatory factor 3 and type I interferons are protective in alcoholic liver injury in mice by way of crosstalk of parenchymal and myeloid cells. Hepatology 53, 649-660.
51.Pupo, E., and Hardy, E. (2009). Complexity and solutions to the isolation problem of Gramnegative lipopolysaccharides'' bacteria molecular species. Biotecnología Aplicada 26.
52.Purohit, V., Bode, J.C., Bode, C., Brenner, D.A., Choudhry, M.A., Hamilton, F., Kang, Y.J., Keshavarzian, A., Rao, R., Sartor, R.B., et al. (2008). Alcohol, intestinal bacterial growth, intestinal permeability to endotoxin, and medical consequences: summary of a symposium. Alcohol 42, 349-361.
53.Rao, R. (2009). Endotoxemia and gut barrier dysfunction in alcoholic liver disease. Hepatology 50, 638-644.
54.Shin, S.M., Yang, J.H., and Ki, S.H. (2013). Role of the Nrf2-ARE pathway in liver diseases. Oxidative medicine and cellular longevity 2013, 763257.
55.Szabo, G. (2010). Alcoholic liver disease and the gut-liver axis. World Journal of Gastroenterology 16, 1321.
56.Szabo, G. (2015). Gut-liver axis in alcoholic liver disease. Gastroenterology 148, 30-36.
57.Tang, W., Jiang, Y.F., Ponnusamy, M., and Diallo, M. (2014). Role of Nrf2 in chronic liver disease. World journal of gastroenterology : WJG 20, 13079-13087.
58.Tietze, F. (1969). Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 27, 502-522.
59.Tsukumo, D.M., Carvalho, B.M., Carvalho-Filho, M.A., and Saad, M.J. (2009). Translational research into gut microbiota: new horizons in obesity treatment. Arq Bras Endocrinol Metabol 53, 139-144.
60.Vine, D.F., Charman, S.A., Gibson, P.R., Sinclair, A.J., and Porter, C.J. (2002). Effect of dietary fatty acids on the intestinal permeability of marker drug compounds in excised rat jejunum. J Pharm Pharmacol 54, 809-819.
61.Waitzberg, D.L., and Torrinhas, R.S. (2009). Fish oil lipid emulsions and immune response: what clinicians need to know. Nutrition in clinical practice : official publication of the American Society for Parenteral and Enteral Nutrition 24, 487-499.
62.Wall, T.L., Thomasson, H.R., Schuckit, M.A., and Ehlers, C.L. (1992). Subjective feelings of alcohol intoxication in Asians with genetic variations of ALDH2 alleles. Alcoholism, clinical and experimental research 16, 991-995.
63.Wang, H., Khor, T.O., Saw, C.L., Lin, W., Wu, T., Huang, Y., and Kong, A.N. (2010). Role of Nrf2 in suppressing LPS-induced inflammation in mouse peritoneal macrophages by polyunsaturated fatty acids docosahexaenoic acid and eicosapentaenoic acid. Molecular pharmaceutics 7, 2185-2193.
64.Wertz, P.W. (2009). Essential fatty acids and dietary stress. Toxicol Ind Health 25, 279-283.
65.Williams, A.J., and Barry, R.E. (1987). Free radical generation by neutrophils: a potential mechanism of cellular injury in acute alcoholic hepatitis. Gut 28, 1157-1161.
66.Wu, D., Wang, X., Zhou, R., and Cederbaum, A. (2010). CYP2E1 enhances ethanol-induced lipid accumulation but impairs autophagy in HepG2 E47 cells. Biochemical and biophysical research communications 402, 116-122.
67.Yan, A.W., and Schnabl, B. (2012). Bacterial translocation and changes in the intestinal microbiome associated with alcoholic liver disease. World J Hepatol 4, 110-118.
68.Yang, M., Gong, S., Ye, S.Q., Lyman, B., Geng, L., Chen, P., and Li, D.Y. (2014). Non-alcoholic fatty liver disease in children: focus on nutritional interventions. Nutrients 6, 4691-4705.
69.Zararsiz, I., Kus, I., Akpolat, N., Songur, A., Ogeturk, M., and Sarsilmaz, M. (2006). Protective effects of omega-3 essential fatty acids against formaldehyde-induced neuronal damage in prefrontal cortex of rats. Cell Biochem Funct 24, 237-244.
70.謝依晴(2010)上皮生長因子改善腸道通透率對於大白鼠酒精性肝臟疾病之影響，台北醫學大學保健營養學系研究所碩士論文
71.廖偉翔(2012)共生質改善酒精餵食大白鼠之腸道菌相及酒精性肝病，台北醫學大學保健營養學系研究所碩士論文
72.李正中(2010)魚油對敗血症小鼠發炎反應及粒線體功能之影響，台北醫學大學保健營養學系研究所碩士論文