簡易檢索 / 詳目顯示

研究生: 高泳詩
Kou, Weng-Si
論文名稱: 海洋深層水對感染胃幽門螺旋桿菌之BALB/c小鼠和AGS細胞可能的預防與治療效用
The preventive and therapeutic effect of deep sea water on Helicobacter Pylori-infected-BALB/c mice and AGS cells
指導教授: 鄭劍廷
Chien, Chiang-Ting
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2018
畢業學年度: 106
語文別: 中文
論文頁數: 91
中文關鍵詞: 幽門螺旋桿菌海洋深層水胃炎
英文關鍵詞: Helicobacter pylori, Deep sea water, Gastritis
DOI URL: http://doi.org/10.6345/THE.NTNU.SLS.001.2018.D01
論文種類: 學術論文
相關次數: 點閱:117下載:0
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報
  • 幽門螺旋桿菌透過感染胃黏膜來引起長期胃發炎,過去已證實其感染與胃潰瘍、十二指腸潰瘍、胃癌等疾病發生有關。海洋深層水富含微量營養素(Micronutrients)與鈣、鎂等礦物質(Minerals),且具有極少病原菌與乾淨等特性。本篇研究探討台灣東部抽取之濃縮海洋深層水之抑菌能力,並使用人類胃癌細胞株(AGS cell)和BALB/c品系小鼠進行感染實驗,來探討給予海洋深層水是否對感染幽門螺旋桿菌具有預防與治療效用。此外,我們利用質譜儀以及真空冷凍乾燥技術來分析海洋深層水內含之未知物,並進一步探討其抑菌能力。
    研究結果顯示不同處理之海洋深層水皆有抑菌效果,抑菌效果逹50%之海洋深層水硬度為1200-3600 ppm,而抑菌效果逹90%之海洋深層水硬度為2400-3600 ppm。因此本篇選用硬度1200 ppm與2400 ppm之海洋深層水進行實驗,從免疫化學結果得知感染後經過海洋深層水處理之細胞相對感染組其細胞凋亡、發炎性細胞凋亡都明顯下降,細胞自噬作用則有增加之趨勢。而經過海洋深層水預防及治療之受感染小鼠相對感染組其細胞凋亡、發炎性細胞凋亡以及氧化壓力均明顯下降,細胞自噬作用則上升。另外,從質譜圖可以得知海洋深層水除了鈣、鎂等礦物質外,還含有大分子之有機物,且其去除二價離子後仍有抑菌的效果,我們推論海洋深層水的未知有機物具有抑菌之效用。
    綜合以上結果得知,台灣東部抽取之濃縮海洋深層水確實有抑菌之效果,並且對感染幽門螺旋桿菌有預防與治療效用,而達到保護胃黏膜,而海洋深層水之有效抑菌成份未來會進一步探討。希望未來能使用海洋深層水作為天然藥物以取代抗生素療法來預防及治療幽門螺旋桿菌。

    Helicobacter pylori (H. Pylori) infects the gastric mucosa and causes long-term inflammation. Previous studies have demonstrated H. pylori may cause gastric ulcer, duodenal ulcer and gastric cancer. Deep sea water (DSW) is rich in micronutrients, minerals such as calcium and magnesium ions and high purity, but less in pathogen.
    This study investigated the antibacterial activity of different types of refined DSW, gathered from eastern Taiwan, and the prevention and therapeutic effect of DSW on H. pylori-infected-AGS cells and H. pylori-infected-BALB/c mice. We further used a mass spectrometer and freeze-drying to analyze the effective antimicrobial composition of DSW.
    The results showed that different types of refined DSW all had antibacterial effects, to reach 50% of antibacterial effects, the hardness of DSW was between 1200-3600 ppm, while achieving 90% of antibacterial effects, the hardness went up to 2400-3600 ppm. Therefore, we used DSW1200 (hardness: 1200 ppm) and DSW2400 (hardness: 2400 ppm) for both cells and animal treatments. The immunochemical tests indicated that DSW-treated H. pylori-infected AGS cells, DSW-pretreated H. pylori-infected mice and DSW-treated H. pylori-infected mice all significantly decreased the expression of apoptosis, pyroptosis and oxidative stress, but increased autophagy expression. Furthermore, it was found that DSW contained massive organic matters by using a mass spectrometer. Another result showed that EDTA in DSW to chelate divalent metal ions still had antibacterial effects. Based on these data, we suggested the antibacterial effective compounds in DSW may be an unknown organic matter, not calcium or magnesium.
    Based on the above results, the refined DSW extracted from eastern Taiwan has the ability of preventive, therapeutic and protective effects on H. pylori infection. The evidence revealed the antibacterial effects of DSW are derived from unknown compounds, but not associated with calcium and magnesium ions. We wish to use DSW as a natural medical alternative of antibiotics to prevent and cure H. pylori infection.

    目錄 摘要 7 Abstract 9 縮寫表 11 第一章 緒論 Introduction 13 1. 幽門螺旋桿菌 14 1.1 幽門螺旋桿菌與消化道疾病之關聯 14 1.2 幽門螺旋桿菌之致病機轉 14 1.3 幽門螺旋桿菌之檢測與療法 16 1.4 幽門螺旋桿菌之另類療法 18 2. 海洋深層水 19 2.1 海洋深層水之特色 19 2.2 海洋深層水之應用 21 第二章 研究材料與方法 Materials and Methods 22 1. 海洋深層水 23 1.1 海洋深層水來源 23 1.2 海洋深層水成份分析 23 2. 幽門螺旋桿菌 24 2.1 幽門螺旋桿菌菌液 24 2.2 海洋深層水抑菌實驗 24 3. 細胞感染實驗 25 4. 動物實驗 25 4.1 動物感染 25 4.2 胃部檢體之細菌培養檢測 28 4.3 組織切片染色觀察(HE Stain) 29 5. 免疫化學分析 30 5.1 細胞免疫化學染色(Immunocytochemistry stain, ICC stain) 30 5.2 組織免疫化學染色(Immunohistochemistry stain, IHC stain) 30 5.3 西方墨點法(Western Blot) 31 5.4 細胞激素蛋白表達微陣列(Cytokine Array) 35 5.5 組織氧化壓力、發炎性細胞凋亡(Pyroptosis)、細胞凋亡(Apoptosis)、自噬作用(Autophagy)檢測 35 5.6 末端脫氧核苷酸轉移酶脫氧尿苷三磷酸切口末端標記(Terminal deoxynucleotidyl transferase dUTP nick end labeling assay, TUNEL assay) 36 6. 資料分析 36 第三章 研究結果 Results 37 1. 海洋深層水的分析 38 2. 海洋深層水對幽門螺旋桿菌感染的治療效益 38 2.1 感染幽門螺旋桿菌的AGS細胞經海洋深層水處理後細胞凋亡的情況 38 2.2 感染幽門螺旋桿菌的AGS細胞經海洋深層水處理後細胞自噬的情況 39 2.3 感染幽門螺旋桿菌的AGS細胞經海洋深層水處理後發炎性細胞凋亡的情況 39 2.4 BALB/c小鼠感染幽門螺旋桿菌後飲用海洋深層水其胃部細胞凋亡的情況 40 2.5 BALB/c小鼠感染幽門螺旋桿菌後飲用海洋深層水其胃部細胞自噬的情況 40 2.6 BALB/c小鼠感染幽門螺旋桿菌後飲用海洋深層水其胃部發炎性細胞凋亡的情況 40 2.7 BALB/c小鼠感染幽門螺旋桿菌後飲用海洋深層水其胃部氧化壓力的情況 41 2.8 BALB/c小鼠感染幽門螺旋桿菌後飲用海洋深層水其胃部白血球浸潤堆積情況 41 3. 海洋深層水對幽門螺旋桿菌感染的預防效益 42 3.1 BALB/c小鼠感染幽門螺旋桿菌前飲用海洋深層水其胃部細胞凋亡的情況 42 3.2 BALB/c小鼠感染幽門螺旋桿菌前飲用海洋深層水其胃部細胞自噬的情況 42 3.3 BALB/c小鼠感染幽門螺旋桿菌前飲用海洋深層水其胃部發炎性細胞凋亡的情況 43 3.4 BALB/c小鼠感染幽門螺旋桿菌前飲用海洋深層水其胃部氧化壓力的情況 43 3.5 BALB/c小鼠感染幽門螺旋桿菌前飲用海洋深層水其胃部白血球浸潤堆積情況 43 3.6 BALB/c小鼠感染幽門螺旋桿菌前飲用海洋深層水其胃部細胞激素蛋白表達微陣列分析 44 4. 進一步探討海洋深層水的成份及性質 45 第四章 討論 Discussion 47 1. 海洋深層水抑菌性探討 48 2. 海洋深層水治療及預防效益 49 3 幽門螺旋桿菌不同程度感染所引發之免疫反應 51 第五章 結論 Conclusion 52 圖表 Figures and Tables 54 參考文獻 86

    1. Perez‐Perez, G. I., Rothenbacher, D., & Brenner, H. (2004). Epidemiology of Helicobacter pylori infection. Helicobacter, 9(s1), 1-6.
    2. Peek, R. M., & Blaser, M. J. (2002). Helicobacter pylori and gastrointestinal tract adenocarcinomas. Nature Reviews Cancer, 2(1), 28-37.
    3. Ghiara, P., Marchetti, M., Blaser, M. J., Tummuru, M. K., Cover, T. L., Segal, E. D., ... & Rappuoli, R. (1995). Role of the Helicobacter pylori virulence factors vacuolating cytotoxin, CagA, and urease in a mouse model of disease. Infection and immunity, 63(10), 4154-4160.
    4. Amieva, M. R., & El–Omar, E. M. (2008). Host-bacterial interactions in Helicobacter pylori infection. Gastroenterology, 134(1), 306-323.
    5. Ha, N. C., Oh, S. T., Sung, J. Y., Cha, K. A., Lee, M. H., & Oh, B. H. (2001). Supramolecular assembly and acid resistance of Helicobacter pylori urease. Nature Structural & Molecular Biology, 8(6), 505-509.
    6. Eaton, K. A., Brooks, C. L., Morgan, D. R., & Krakowka, S. (1991). Essential role of urease in pathogenesis of gastritis induced by Helicobacter pylori in gnotobiotic piglets. Infection and immunity, 59(7), 2470-2475.
    7. Handa, O., Naito, Y., & Yoshikawa, T. (2010). Helicobacter pylori: a ROS-inducing bacterial species in the stomach. Inflammation Research, 59(12), 997-1003.
    8. Yang, J. C., Yang, H. C., Shun, C. T., Wang, T. H., Chien, C. T., & Kao, J. Y. (2013). Catechins and sialic acid attenuate helicobacter pylori-triggered epithelial caspase-1 activity and eradicate helicobacter pylori infection. Evidence-Based Complementary and Alternative Medicine, 2013.
    9. Crabtree, J. E., Covacci, A., Farmery, S. M., Xiang, Z., Tompkins, D. S., Perry, S., ... & Rappuoli, R. (1995). Helicobacter pylori induced interleukin-8 expression in gastric epithelial cells is associated with CagA positive phenotype. Journal of clinical pathology, 48(1), 41-45.
    10. Blaser, M. J., Perez-Perez, G. I., Kleanthous, H., Cover, T. L., Peek, R. M., Chyou, P. H., ... & Nomura, A. (1995). Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach. Cancer research, 55(10), 2111-2115.
    11. Parsonnet, J., Friedman, G. D., Orentreich, N., & Vogelman, H. (1997). Risk for gastric cancer in people with CagA positive or CagA negative Helicobacter pylori infection. Gut, 40(3), 297-301.
    12. Atherton, J. C., Cao, P., Peek, R. M., Tummuru, M. K., Blaser, M. J., & Cover, T. L. (1995). Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori association of specific vacA types with cytotoxin production and peptic ulceration. Journal of Biological Chemistry, 270(30), 17771-17777.
    13. Kuck, D., Kolmerer, B., Iking-Konert, C., Krammer, P. H., Stremmel, W., & Rudi, J. (2001). Vacuolating cytotoxin of Helicobacter pylori induces apoptosis in the human gastric epithelial cell line AGS. Infection and immunity, 69(8), 5080-5087.
    14. Slomiany, B. L., & Slomiany, A. (1992). Mechanism of Helicobacter pylori pathogenesis: focus on mucus. Journal of clinical gastroenterology, 14, S114-21.
    15. Tannaes, T., Bukholm, I. K., & Bukholm, G. (2005). High relative content of lysophospholipids of Helicobacter pylori mediates increased risk for ulcer disease. FEMS Immunology & Medical Microbiology, 44(1), 17-23.
    16. Gisbert, J. P., De La Morena, F., & Abraira, V. (2006). Accuracy of monoclonal stool antigen test for the diagnosis of H. pylori infection: a systematic review and meta-analysis. The American journal of gastroenterology, 101(8), 1921-1930.
    17. Konstantopoulos, N., Rüssmann, H., Tasch, C., Sauerwald, T., Demmelmair, H., Autenrieth, I., & Koletzko, S. (2001). Evaluation of the Helicobacter pylori stool antigen test (HpSA) for detection of Helicobacter pylori infection in children. The American journal of gastroenterology, 96(3), 677-683.
    18. Cardinali, L. D. C. C., Rocha, G. A., Rocha, A. M. C., de Moura, S. B., de Figueiredo Soares, T., Esteves, A. M. B., ... & Ferreira, A. (2003). Evaluation of [13C] urea breath test and Helicobacter pylori stool antigen test for diagnosis of H. pylori infection in children from a developing country. Journal of clinical microbiology, 41(7), 3334-3335.
    19. Ricci, C., Holton, J., & Vaira, D. (2007). Diagnosis of Helicobacter pylori: invasive and non-invasive tests. Best practice & research Clinical gastroenterology, 21(2), 299-313.
    20. Gene, E., Calvet, X., Azagra, R., & Gisbert, J. P. (2003). Triple vs. quadruple therapy for treating Helicobacter pylori infection: a meta‐analysis. Alimentary pharmacology & therapeutics, 17(9), 1137-1143.
    21. Kawabata, H., Habu, Y., Tomioka, H., Kutsumi, H., Kobayashi, M., Oyasu, K., ... & Kimoto, K. (2003). Effect of different proton pump inhibitors, differences in CYP2C19 genotype and antibiotic resistance on the eradication rate of Helicobacter pylori infection by a 1‐week regimen of proton pump inhibitor, amoxicillin and clarithromycin. Alimentary pharmacology & therapeutics, 17(2), 259-264.
    22. Lambert, J. R., & Midolo, P. (1997). The actions of bismuth in the treatment of Helicobacter pylori infection. Alimentary pharmacology & therapeutics, 11(S1), 27-33.
    23. Szajewska, H., Horvath, A., & Piwowarczyk, A. (2010). Meta‐analysis: the effects of Saccharomyces boulardii supplementation on Helicobacter pylori eradication rates and side effects during treatment. Alimentary pharmacology & therapeutics, 32(9), 1069-1079.
    24. De Francesco, V., Giorgio, F., Hassan, C., Manes, G., Vannella, L., Panella, C., ... & Zullo, A. (2010). Worldwide H. pylori antibiotic resistance: a systematic review. Journal of Gastrointestinal & Liver Diseases, 19(4).
    25. Su, P., Li, Y., Li, H., Zhang, J., Lin, L., Wang, Q., ... & Shi, Z. (2013). Antibiotic resistance of Helicobacter pylori isolated in the Southeast Coastal Region of China. Helicobacter, 18(4), 274-279.
    26. Liou, J. M., Chang, C. Y., Chen, M. J., Chen, C. C., Fang, Y. J., Lee, J. Y., ... & Tseng, C. H. (2015). The primary resistance of Helicobacter pylori in Taiwan after the national policy to restrict antibiotic consumption and its relation to virulence factors—a nationwide study. PLoS One, 10(5), e0124199.
    27. Thung, I., Aramin, H., Vavinskaya, V., Gupta, S., Park, J. Y., Crowe, S. E., & Valasek, M. A. (2016). the global emergence of Helicobacter pylori antibiotic resistance. Alimentary pharmacology & therapeutics, 43(4), 514-533.
    28. Tong, J. L., Ran, Z. H., Shen, J., Zhang, C. X., & Xiao, S. D. (2007). Meta‐analysis: the effect of supplementation with probiotics on eradication rates and adverse events during Helicobacter pylori eradication therapy. Alimentary pharmacology & therapeutics, 25(2), 155-168.
    29. Zhang, L., Ma, J., Pan, K., Go, V. L. W., Chen, J., & You, W. C. (2005). Efficacy of Cranberry Juice on Helicobacter pylori Infection: a Double‐Blind, Randomized Placebo‐Controlled Trial. Helicobacter, 10(2), 139-145.
    30. Hwang, H. S., Kim, H. A., Lee, S. H., & Yun, J. W. (2009). Anti-obesity and antidiabetic effects of deep sea water on ob/ob mice. Marine biotechnology, 11(4), 531.
    31. Nakasone, T., & Akeda, S. (1999, November). The application of deep sea water in Japan. In Proc 28th UJNR Aquac Panel Symp, UJNR Technical Report (No. 28, pp. 69-75).
    32. Tsuchiya, Y., Watanabe, A., Fujisawa, N., Kaneko, T., Ishizu, T., Fujimoto, T., ... & Yamamoto, M. (2004). Effects of desalted deep seawater on hematologic and blood chemical values in mice. The Tohoku journal of experimental medicine, 203(3), 175-182.
    33. Liang, W. D., Tang, T. Y., Yang, Y. J., Ko, M. T., & Chuang, W. S. (2003). Upper-ocean currents around Taiwan. Deep Sea Research Part II: Topical Studies in Oceanography, 50(6), 1085-1105.
    34. Liu, K. K., Peng, T. H., Shaw, P. T., & Shiah, F. K. (2003). Circulation and biogeochemical processes in the East China Sea and the vicinity of Taiwan: an overview and a brief synthesis.
    35. Fu, Z. Y., Yang, F. L., Hsu, H. W., & Lu, Y. F. (2012). Drinking Deep Seawater Decreases Serum Total and Low-Density Lipoprotein–Cholesterol in Hypercholesterolemic Subjects. Journal of medicinal food, 15(6), 535-541.
    36. Sheu, M. J., Chou, P. Y., Lin, W. H., Pan, C. H., Chien, Y. C., Chung, Y. L., ... & Wu, C. H. (2013). Deep sea water modulates blood pressure and exhibits hypolipidemic effects via the AMPK-ACC pathway: an in vivo study. Marine drugs, 11(6), 2183-2202.
    37. Liu, H. Y., Liu, M. C., Wang, M. F., Chen, W. H., Tsai, C. Y., Wu, K. H., ... & Deng, W. P. (2013). Potential osteoporosis recovery by deep sea water through bone regeneration in SAMP8 mice. Evidence-Based Complementary and Alternative Medicine, 2013.
    38. Lee, D. H., Kim, S., & Nam, K. S. (2014). Protective effects of deep sea water against doxorubicin induced cardiotoxicity in H9c2 cardiac muscle cells. International journal of oncology, 45(6), 2569-2575.
    39. Yang, C. C., Yao, C. A., Lin, Y. R., Yang, J. C., & Chien, C. T. (2014). Deep-sea water containing selenium provides intestinal protection against duodenal ulcers through the upregulation of Bcl-2 and thioredoxin reductase 1. PloS one, 9(7), e96006.
    40. Yokota, J., Kitaoka, T., Jobu, K., Takuma, D., Hamada, A., Onogawa, M., ... & Miyamura, M. (2011). Eriobotrya japonica seed extract and deep sea water protect against indomethacin-induced gastric mucosal injury in rats. Journal of natural medicines, 65(1), 9-17.
    41. Kawada, M., & Takeuchi, H. (2013). Antibacterial Activities of Refined Deep Seawater on Helicobacter pylori. Journal of Genetic Syndromes & Gene Therapy, s1. doi: 10.4172/2161-0703.s1-002
    42. Mohd Nani, S. Z., Majid, F. A. A., Jaafar, A. B., Mahdzir, A., & Musa, M. N. (2016). Potential Health Benefits of Deep Sea Water: A Review. Evidence-Based Complementary and Alternative Medicine, 2016.
    43. Xu, H., Chaturvedi, R., Cheng, Y., Bussiere, F. I., Asim, M., Yao, M. D., ... & Moss, S. F. (2004). Spermine Oxidation Induced by Helicobacter pylori Results in Apoptosis and DNA Damage. Cancer research, 64(23), 8521-8525.
    44. Ding, S. Z., Minohara, Y., Fan, X. J., Wang, J., Reyes, V. E., Patel, J., ... & Crowe, S. E. (2007). Helicobacter pylori infection induces oxidative stress and programmed cell death in human gastric epithelial cells. Infection and immunity, 75(8), 4030-4039.
    45. Greenfield, L. K., & Jones, N. L. (2013). Modulation of autophagy by Helicobacter pylori and its role in gastric carcinogenesis. Trends in microbiology, 21(11), 602-612.
    46. Calvino, F. M., & Parra, C. T. (2010). H. pylori and mitochondrial changes in epithelial cells. The role of oxidative stress. Revista espanola de enfermedades digestivas: organo oficial de la Sociedad Espanola de Patologia Digestiva, 102(1), 41-50.
    47. Monack, D. M., Mueller, A., & Falkow, S. (2004). Persistent bacterial infections: the interface of the pathogen and the host immune system. Nature Reviews Microbiology, 2(9), 747-765.
    48. Gobert, A. P., & Wilson, K. T. (2016). The immune battle against Helicobacter pylori infection: NO offense. Trends in microbiology, 24(5), 366-376.

    無法下載圖示 本全文未授權公開
    QR CODE