臺灣博碩士論文加值系統

抗癌藥物是常引起系統性免疫抑制及腸道受損，如5-氟尿嘧啶(5-FU)是常見的化療藥物，在臨床上是毒殺腸胃道癌症有較好的治療效果，但常見之副作用為噁心、嘔吐及腹瀉導致體重減輕，嚴重時易引發免疫發炎反應與相關併發症，及抗藥性等問題。化療藥物在對抗癌細胞的同時，也破壞了正常腸道結構，造成腸道黏膜炎，腹瀉等副作用發生，因此在這情況下，發展解決因化學治療製劑產生的副作用是必須的。在過去對於腸胃道疾病的治療，乳酸菌一直被受到注意，因為乳酸菌本身是可促進或減緩腸道的防禦系統功能，如免疫調節作用、免疫減緩作用和免疫排斥反應，且具有抗發炎、調整腸道完整度，以及調控過敏反應等。
因此，在這項實驗中，利用化療藥物5-氟尿嘧啶(5-FU)誘導Caco-2細胞損傷產生發炎反應，並分別以活菌與85℃, 20分鐘的死菌加入於5-FU 誘導Caco-2細胞上的作用，調控發炎反應的能力。初步實驗結果顯示，以2、4小時，MOI=25的活菌與死菌(85℃, 20分鐘)進行毒性分析後，其具有良好生物相容性。最後由基因表現量的結果顯示，經過活菌與死菌經過2小時的作用下，IL-12、TNF-α、MCP-1之mRNA都有明顯向上調控的趨勢；經過4小時的作用，活菌與死菌之間的發炎反應量沒有像在2小時的作用下表現量這麼高，其原因可能是因為在4時間的作用下細胞似乎隨著時間的延長受到不同型式乳酸菌的影響促進的發炎反應趨勢會緩緩向下調控。

Many anti-cancer medicine usually cause system immune inhibition and impaired gut, such as 5-Fluorouracil (5-FU) is a common chemotherapy drug. 5-FU cytotoxicity in clinical gastrointestinal cancers is better therapeutic. It still has common side effects which are nausea, vomiting and diarrhea lead to body weight lose. Chemotherapy drugs also damage the normal intestinal structure, causing intestinal mucous, diarrhea and other side effects occurred in the fight against cancer at the same time. It is impassion serious inflammation and immune-related complications and drug resistance. It’s necessary to reduce side effect of immune inhibition. Therefore, in this case, it’s necessary that development to solve side effects of chemotherapy drugs. In the past, for the treatment of gastrointestinal diseases that lactobacilli has been received attention, because lactobacilli can promote or slow down the intestinal defense system functions, such as immune regulation immune to slow down the role of immune rejection, and has anti-inflammatory, regulate intestinal integrity, and regulation of allergic reactions.
In this study, Caco-2 cells were induced inflammatory response by 5-FU and were treated with the live LGG or heat killed LGG (at 85℃ for 20 min) thereafter the effect to regulate inflammatory responses. These data showed the live LGG and heat killed LGG at an MOI of 25 for 2 and 4 hours treating times that exerted good biocompatibility via the cytotoxicity test. The results of gene expression indicated that after 2 hours live LGG and heat killed LGG treatment, the mRNA expression of IL-12, TNF-α, MCP-1 were significantly up-regulation; after 4 hours treatment, expression of these inflammatory mRNA were lower than 2 hours treatment. We considered, the host cells may be influenced by different types of LGG under 4 hours treatment and inflammatory response was slightly down-regulated.

中文摘要 .i
英文摘要 ii
目錄 .iv
表目錄 .vi
圖目錄 vii
第一章 緒論 1
1.1 前言 1
1.2 實驗動機與目的 2
1.3 實驗設計流程 4
第二章 文獻回顧 5
2.1 腸道結構與功能 5
2.1.1 腸道上皮細胞之生理功能 5
2.1.2 腺窩-絨毛軸 6
2.1.3 大腸直腸癌之發生 6
2.1.4 化療藥物對腸道的影響 7
2.1.5 化療藥物 5-Fluorouracil 8
2.2 人體腸道菌 9
2.2.1 腸道中的細菌數 9
2.2.2 腸道菌相 10
2.2.3 影響腸道菌相因子 10
2.3 益生菌 10
2.3.1 益生菌定義 10
2.3.2 益生菌特性 11
2.3.3 益生菌在腸道中的作用機制 13
2.3.4 益生菌的生理功能 14
2.3.5 益生菌的吸附機制 15
第三章 材料與方法 16
3.1 藥品與試劑 16
3.2 儀器設備 18
3.3 Caco-2細胞培養方法 19
3.3.1 建立Caco-2細胞 19
3.3.2 冷凍細胞活化 20
3.3.3 細胞繼代培養 20
3.3.4 細胞冷凍保存 21
3.3.5 細胞計數 21
3.4 建立化療藥物5-FU誘導Caco-2細胞損傷模式 21
3.4.1 MTT之細胞活性測試 22
3.4.2 化療藥物5-FU誘導Caco-2細胞發炎 (Inflammatory) 22
3.4.3 化療藥物5-FU 誘導腸修復之細胞毒性（Cytotoxicity）
分析 23
3.5 細菌培養方法 24
3.5.1 菌株 24
3.5.2 菌種培養與保存 24
3.5.3 菌株培養基 24
3.5.4 製備不同型式Lactobacillus rhamnosus GG 24
3.5.5 不同型式Lactobacillus rhamnosus GG型態評估 25
3.6 不同型式Lactobacillus rhamnosus GG對5-FU誘導腸修復之
細胞毒性分析 25
3.6.1 Lactate Dehydrogenase（LDH）分析 26
3.7 基因表現之評估 27
3.7.1 RNA之純化 27
3.7.2 反轉錄聚合酶反應（Reverse Transcription-PCR） 27
3.7.3 定量聚合酶鏈鎖反應（Quantifcation Real-Time PCR） 28
3.8 不同型式Lactobacillus rhamnosus GG對5-FU誘導腸發炎
基因表現效應 30
第四章 結果與討論 31
4.1 Caco-2細胞培養方法 31
4.1.1 建立Caco-2細胞 31
4.2 建立化療藥物5-FU誘導Caco-2細胞損傷模型 33
4.2.1 MTT之細胞活性測試 33
4.2.2 化療藥物5-FU誘導Caco-2細胞發炎 (Inflammatory) 34
4.2.3 化療藥物5-FU 誘導腸修復之細胞毒性（Cytotoxicity）
分析 35
4.3 細菌培養方法 36
4.3.1 製備不同型式Lactobacillus rhamnosus GG 36
4.3.2 不同型式之Lactobacillus rhamnosus GG型態評估 38
4.4 不同型式Lactobacillus rhamnosus GG對5-FU誘導腸修復之
細胞毒性（Cytotoxicity）分析 40
4.4.1 Lactate Dehydrogenase（LDH）分析 40
4.5 不同型式Lactobacillus rhamnosus GG對5-FU誘導腸發炎
基因表現之評估 42
第五章 結論 46
參考文獻 47

1.行政院衛生署，中華民國九十九年台灣地區十大死因統計結果, 2010
2.Muehlbauer, P. M., Thorpe, D., Davis, A., Drabot, R., Rawlings, B. L., Kiker, E. “Putting evidence into practice: evidence-based interventions to prevent, manage, and treat chemotherapy- and radiotherapy-induced diarrhea.” Clin J Oncol Nurs vol.13, 2009, pp.336-341.
3.Benson, A. B., Ajani, J. A., Catalano, R. B., Engelking, C., Kornblau, S. M., Martenson, J. A., Jr., McCallum, R., Mitchell, E. P., O''Dorisio, T. M., Vokes, E. E., Wadler, S. “Recommended guidelines for the treatment of cancer treatment-induced diarrhea.” J Clin Oncol, vol.22, 2004, pp.2918-2926.
4.Kornblau, S., Benson, A. B., Catalano, R., Champlin, R. E., Engelking, C., Field, M., Ippoliti, C., Lazarus, H. M., Mitchell, E., Rubin, J., Stiff, P. J., Vokes, E., Wadler, S. “Management of cancer treatment-related diarrhea. Issues and therapeutic strategies.” J Pain Symptom Manage, vol.19, 2000, pp.118-129.
5.Stringer, A. M., Gibson, R. J., Bowen, J. M., Logan, R. M., Yeoh, A. S., Keefe, D. M. “Chemotherapy-induced mucositis: the role of gastrointestinal microflora and mucins in the luminal environment.” J Support Oncol, vol.5, 2007, pp.259-267.
6.Bowen, J. M., Stringer, A. M., Gibson, R. J., Yeoh, A. S., Hannam, S., Keefe, D. M. “VSL#3 probiotic treatment reduces chemotherapy-induced diarrhea and weight loss.” Cancer Biol Ther, vol.6, 2007, pp.1449-1454.
7.Von Bultzingslowen, I., Adlerberth, I., Wold, A. E., Dahlen, G., Jontell, M. “Oral and intestinal microflora in 5-fluorouracil treated rats, translocation to cervical and mesenteric lymph nodes and effects of probiotic bacteria.” Oral Microbiol Immunol, vol.18, 2003, pp.278-284.
8.Duffy, L. C. “Interactions mediating bacterial translocation in the immature intestine.” J Nutr, vol.130, 2000, pp.432-436.
9.Mariela, L., Nan L., Jasmeet, K., Michael, R., Josef, N. “Live and Ultraviolet-Inactivated Lactobacillus Rhamnosus GG Decrease Flagellin-Induced Interleukin-8 Production in Caco-2 Cells.” J. Nutr., vol.138, 2008, pp.2264–2268.
10.Rana, A., Michel, B., Thomas, M. “Mechanism of cytokine modulation of epithelial tight junction barrier.” Frontiers in Bioscience, vol.14, 2009, pp.2765-2778
11.Kevin, A., Melanie, G., Yu, J., Jenny, W, Philip, M. “Lactobacillus rhamnosus GG attenuates interferon-{gamma} and tumor necrosis factor-{alpha}-induced barrier dysfunction and pro-inflammatory signalling.” Microbiology Papers in Press, 2010.
12.黎莉婷，建立具有極化且緊密連結組織結構之腸道上皮細胞單層膜，國立臺北科技大學，2009
13.Compton, C. C., and Greene, F. L. 2004. “The staging of colorectal cancer: 2004 and beyond.”CA Cancer J Clin ,pp.295-308.
14.Kohnoe, S., Kakeji, Y., Maehara, Y. “Progress in adjuvant therapy for colorectal cancer.” Gan To Kagaku Ryoho, vol.29, 2002, pp.2488-2497.
15.Frederiksen, C.M., Knudsen, S., Laurberg, S., TF, O.R. “Classification of Dukes'' B and C colorectal cancers using expression arrays.” J Cancer Res Clin Oncol.
16.Biswal, B.M., Sain, A.H., Othman, N.H., Baba, A. “Adjuvant treatment in colorectal cancer. Experience from a referral center in eastern peninsular Malaysia.” Trop Gastroenterol, vol.23, 2002, pp.134-137.
17.Keating, J.P., Pater, P., Lolohea, S., Wickremesekera, J.K. “The epidemiology of colorectal cancer: what can we learn from the New Zealand Cancer Registry?” N Z Med J, vol.116, 2003, pp.437-437.
18.Tokudome, S., Nagaya, T., Okuyama, H., Tokudome, Y., Imaeda, N., Kitagawa, I., Fujiwara, N., Ikeda, M., Goto, C., Ichikawa, H., Kuriki, K., Takekuma, K., Shimoda, A., Hirose, K., Usui, T. “Japanese versus Mediterranean Diets and Cancer.” Asian Pac J Cancer Prev, vol.1, 2000, pp.61-66.
19.Dray, X., Boutron-Ruault, M.C., Bertrais, S., Sapinho, D., Benhamiche-Bouvier, A.M., Faivre, J. “Influence of dietary factors on colorectal cancer survival.” Gut, vol.52, 2003, pp.868-873.
20.Hill, M. “Dietary fibre and colon cancer: where do we go from here?” Proc Nutr Soc, vol.62, 2003, pp.63-65.
21.Hobday, T.J., Erlichman, C. “Adjuvant therapy of colon cancer: a review.” Clin Colorectal Cancer, vol.1, 2002, pp.230-236.
22.Kohnoe, S., Kakeji, Y., Maehara, Y. “Progress in adjuvant therapy for colorectal cancer.” Gan To Kagaku Ryoho, vol.29, 2002, pp.2488-2497.
23.Partyka, S., Ajani, J. “Chemotherapy of Colorectal Cancer.” Curr Treat Options Gastroenterol, vol.2, 1999, pp.38-48.
24.Kobayashi, T., Sawa, H., Morikawa, J., Zhang, W. Shiku, H. “Bax induction activates apoptotic cascade via mitochondrial cytochrome c release and Bax overexpression enhances apoptosis induced by chemotherapeutic agents in DLD-1 colon cancer cells.” Jpn J Cancer Res, vol.91, 2000, pp.1264-1268.
25.Uchida, H., Shinoura, N., Kitayama, J., Watanabe, T., Nagawa, H., Hamada, H. “5-Fluorouracil efficiently enhanced apoptosis induced by adenovirus-mediated transfer of caspase-8 in DLD-1 colon cancer cells.” J Gene Med,vol.5, 2003, pp.287-299.
26.Gianola, F.J., Sugarbaker, P.H., Barofsky, I., White, D.E. Meyers, C.E. “Toxicity studies of adjuvant intravenous versus intraperitoneal 5-FU in patients with advanced primary colon or rectal cancer.” Am J Clin Oncol, vol.9, 1986, pp.403-410.
27.Shehata, N., Pater, A., Tang, S.C. “Prolonged severe 5-fluorouracil-associated neurotoxicity in a patient with dihydropyrimidine dehydrogenase deficiency.” Cancer Invest, vol.17, 1999, pp.201-205.
28.Peter G J, Jansen G, Schilsky R L et al ”Principles of antiplastic drug development and pharmacology.” Marcel Dekker. 1996. pp.543-585.
29.Montfort WR, Weichsel A “Thymidylate synthase: structure, inhibition, and strained conformations during catalysis.” Pharmacol Ther. vol.76, 1997. pp.29-43.
30.Sobrero A F, Aschele C, Bertino J R. “Fluorouracil in colorectal cancer- a tale of two drugs:implications for biochemical modulation.” J Clin Oncol, vol.15 1997 pp.368-381.
31.Mader R M, Muller M, Steger G G. “Resistance to 5-fluorouracil.” Gen Pharmacol vol31,1998. pp.661-666.
32.Pinedo H. M., Peters GF. “Fluorouracil: biochemistry and pharmacology.” J Clin Oncol. vol.6, 1988, pp.1653-1664.
33.Longley D. B, Harkin DP, Johnston PG “5-fluorouracil: mechanisms of action and clinical strategies.” Nat Rev Cancer. vol.3, 2003, pp.330-338.
34.Diasio R B.,Harris BE. “Clinical pharmacology of 5-fluorouracil.” Clin Pharmacokinet. vol.16, 1989, pp.215-237.
35.Carreras C. W., Santi DV. “The catalytic mechanism and structure of thymidylate synthase.” Annu Rev Biochem. vol.64, 1995, pp.721-762.
36.丁懷謙，益生菌與胃腸保健功效。食品工業月刊。2000年，第32期(10)第1-7頁。
37.Fooks, L. J., R. Fuller, and G. R. Gibson. “Prebiotics, probiotics and human gut microbiology.” Int. Dairy J. vol.9, 1999, pp.53-61.
38.Steer, T., H. Carpenter, K. Tuohy, G. R. Gibson. “Perspective on the role of the human gut microbiota and its modulation by pro- and prebiotics.” Nutr. Res. Rev. vol.13, 2000, pp.229-254.
39.Ziemer, C. J., G. R. Gibson. “An overview of probiotics, prebiotics and synbiotics in the functional food concept: perspectives and future strategies.” Int. Dairy J. vol.8, 1998, pp.473-479.
40.Ostlie, H. M., M. H. Helland, J. A. Narvhus. “Growth and metabolism of selected strains of probiotic bacteria in milk.” Int. J. Food Microbiol. vol.87,2003, pp.17-27.
41.Pinto, M., Robine-Leon, S., Appay, M. D., Kedinger, M., Triadou, N. ,Dussaulx, E., Lacroix, B., Simon-Assmann, P., Haffen, K., Fogh, J. andZweibaum, A. “Enterocyte-like differentiation and polarization of thehuman colon carcinoma cell line Caco-2 in culture.” Biol. Cell. vol.47, 1983, pp.323-330.
42.Kociubinski, G., P. Perez, M. Anon, and G. De Antoni. “Amethod of screening of highly inhibitory lactic acid bacteria.” J. Food Prot. 1996, vol.59, pp.1-8.
43.Linggren, S. E., and W. J. “Dobrogosz. Antagonistic activities of lactic acid bacteria in food and feed dermentations.” FEMS Microbiol. Rev. vol.87, 1990, pp.149-163.
44.Motlagh, A. M., M. C. Johnson, and B. Ray. “Viability loss of foodborne pathogens by starter culture metabolites.” J. Food Protect. 1991, pp.873-878.
45.Bruno, M. E. C., and T. J. Montville. “Common mechanistic action of bacteriocins from lactic acid bacteria.” Appl. Environ. Microbiol. vol.59, 1993, pp.3003-3010.
46.Lee, Y.K. and S. Salminen. “The coming of age of probiotics.” Trends Food Sci. Technol., vol.6, 1995, pp.241-245.
47.Schuler-Malyoth R, Ruppert A, Muller F. “The microorga-nisms of the bifidus group (Lactobacillus bifidus). 1. Historical review, nutritional, physiological and therapeutic aspects, mor-phology, culture procedures and taxonomy.” Mitch-wissenschaft. vol.23, 1968, pp.35 - 360.
48.楊媛絢，「原生保健性菌種與益助性生質之應用」，食品工業， 1998年，第30期，第11-22頁。
49.Arunachalam KD., “Role of bifidobacteria in nutrition, medicine and technology,” Nutr Res, vol.19, 1999, pp. 1559-1597 .
50.Kaur, N., Wohlhueter, A.L., Halvorsen, S.W. “Activation and inactivation of signal transducers and activators of transcription by ciliary neurotrophic factor in neuroblastoma cells.” Cell. Signal. vol.14, 2002, pp.419–429.
51.Fooks LJ., Fuller R. and Gibson GR., “Prebiotics, probiotics and human gut microbiology,” Int. Dairy J., vol. 9, 1999, pp. 53-61.
52.Helander IM, von Wright A, Mattila-Sandholm TM. “Potential of lactic acid bacteria and novel antimicrobials against Gram-negative bacteria.” Trends Food Sci. Technol. vol.8, 1997, pp.146-150.
53.McNaught, C.E., MacFie, J. “Probiotics in clinical practice: a critical review of the evidence.” Nutrition Research, vol.21, 2001, pp.343–353.
54.Roos, N.M. and M.B. Katan. “Effects of probiotic bacteria on diarrhea, lipid metabolism and carcinogenesis: A review of papers published between 1988 and 1998.” Am. J. Clin. Nutr., vol.71, 2000, pp.405-411.