幽門螺旋桿菌(Helicobacter pylori, H. pylori)為一種彎曲桿狀，存在於胃腸道黏膜上的革蘭氏陰性微需氧菌，主要寄居在胃腸道黏膜上，感染者會增加罹患胃癌的風險。為了避免幽門桿菌導致的胃癌風險，目前最常用的療程為抗生素加上制酸劑，將細菌根除並減低胃黏膜的傷害。然而，近年抗生素抗藥性導致療程失敗，以及人體微生物菌叢議題的崛起，使人們開始研究益生菌對幽門桿菌的影響。然而，益生菌的測試，用動物實驗十分耗時，在個體間也有極大的差異，因此開發一個模擬平台來探討益生菌對幽門螺旋桿菌的抑制作用，有極大的實用性。
本研究使用3D列印技術列印培養盒，培養盒內表面經過大氣電漿噴塗增加親水性，使混和尿素及胃黏膜蛋白的定植層可順利黏附。接續將培養盒置於含有模擬胃液之動態培養系統，並在不同條件下觀察生長情形，找出最有利對於幽門螺旋桿菌生長條件。接著在模擬人體胃部感染幽門桿菌的環境條件下，測試羅伊氏桿菌(Lactobacillus reuteri)投入後對幽門螺旋桿菌的抑制情況。
研究結果顯示，經過電漿表面改質後3D培養盒之mucin厚度為0.118 mm，與未經處理的培養盒的mucin厚度為0.097 mm，而增加17.80%。在添加上蓋與胃蛋白酶(Pepsin)，以及尿素的組別中H. pylori，菌量有60倍的成長(pH=3，有厭氧)，在添加上蓋與Pepsin，以及尿素的組別中Lactobacillus reuteri，菌量有48倍的成長(pH=3，無厭氧)，結果顯示此平台可模擬了幽門螺旋桿菌植入人體胃部時幽門螺旋桿菌的生長情況。在初步共同培養的過程中，雖然在單次添加Lactobacillus reuteri對H. Pylori生長沒有明顯的抑制效果，但是在新的實驗結果中，投入Reuterin對H. pylori的抑制試驗中之生長評估，添加不同濃度的Reuterin跟H. pylori進行培養，會讓抑制H. Pylori的生長， Reuterin加的量越多，會對H. pylori有更明顯的抑制效果。之後也可以利用培養平台測試多次添加Lactobacillus reuteri，或結合抗生素或其他益生菌對模擬胃中幽門螺旋桿菌的抑制作用。預期此幽門螺旋桿菌體外試驗平台，可運用在檢測日後市面上之藥物、健康食品等在模擬人體胃中對幽門螺旋桿菌的生長抑制效果。

Helicobacter pylori (H.pylori) is the curved rod-shaped Gram-negative microaerobic bacteria that mainly inhabits in the mucosa of the gastrointestinal tract. People infected with Helicobacter pylori may develop chronic gastritis after Helicobacter pylori infection, which increases the risk of gastric cancer. The current common treatment for Helicobacter pylori is the combination of proton pump blockers and antibiotics. Recently, due to the bacterial resistance causing treatment failure and the rise of gut microbiota issue, the influence of probiotics to H. pylori became a subject worth studying. However, there are deviations from the effect of probiotics between individules, and animal testing is time consuming. Therefore, developing a platform to simulate the interaction between probiotics and H. pylori is essential and pratical.
In this study, 3D printing technology was used to print the culture box and set up a simulating human stomach environment. After increasing hydrophilicity by atmospheric plasma spraying, the urea and gastric mucosal protein coating to the culture box is more easily. In order to find out the most favorable conditions, the survival rate of H. pylori in different environment of low pH gastric acid is evaluated. Then Lactobacillus reuteri is added to simulate human stomach environmental platform to see its imapact on the growth of Helicobacter pylori.
The results of the study showed that the mucin thickness of the 3D culture box after plasma surface modification was 0.118 mm, 17.80% higher than and that of the untreated one with 0.097 mm. When cultured under the condition with lid, pepsin, and urea, H. pylori and Lactobacillus reuteri exhibited a 60-fold (pH=3, anaerobic), and 48-fold growth in the bacterial population compared with the control groups cultured with mucin only. The results show that this platform can simulate the growth of Helicobacter pylori when it is implanted in the human stomach. During the initial co-cultivation process, a single addition of Lactobacillus reuteri had no obvious inhibitory effect on the growth of H. pylori. A single addition of Lactobacillus reuteri had no obvious inhibitory effect on the growth of H. Pylori. Another experiment was then set up, in which Reuterin was added to H. pylori to evaluate the inhibitory effect on H. Pylori. growth. The result indicated that Reuterin did inhibit the growth of H. Pylori, i.e. the more the Reuterin added, the more obvious the inhibitory effect on H. pylori. However, the culture platform can be used to test multiple additions of Lactobacillus reuteri, or combined with antibiotics or other probiotics to study the inhibitory effect against H.pylori in the stimulated stomach condition. It is expected that this Helicobacter pylori in vitro test platform can be used to detect the growth inhibitory effects of commercial drugs and health supplements against Helicobacter pylori in simulated human stomach condition in the future.

1. Muran, L.I., et al., Study on the relationship between the pathologic change of chronic atrophic gastritis and helicobacer pylori. Tianjin Medical Journal, 2015(1): p. 54-56.
2. Thamphiwatana, S., et al., In vivo treatment of <em>Helicobacter pylori</em> infection with liposomal linolenic acid reduces colonization and ameliorates inflammation. 2014. 111(49): p. 17600-17605.
3. Tripathi, G.K., S. Singh, and G. Nath, Formulation and In-vitro Evaluation of pH-Sensitive Oil Entrapped Polymeric Blend Amoxicillin Beads for the Eradication of Helicobacter pylori. Iranian journal of pharmaceutical research : IJPR, 2012. 11(2): p. 447-455.
4. Ji, J. and H. Yang, Using probiotics as supplementation for Helicobacter pylori antibiotic therapy. International journal of molecular sciences, 2020. 21(3): p. 1136.
5. Edermaniger, L., The Facts On Taking Probiotics With Antibiotics, And Antibiotics With Food. Atlas blog (2020) March 19.
6. Smith, S.M., C. O’Morain, and D. McNamara, Antimicrobial susceptibility testing for Helicobacter pylori in times of increasing antibiotic resistance. World Journal of Gastroenterology: WJG, 2014. 20(29): p. 9912.
7. Schistosomes, L.F., Helicobacter pylori. Vol. 61. International Agency for Research on Cancer. World Health Organization, 1994.
8. Rugge, M., et al., Gastric Cancer as Preventable Disease. Clin Gastroenterol Hepatol, 2017. 15(12): p. 1833-1843.
9. Krueger, S., A. Roessner, and D. Kuester, Murine models of H. pylori-induced gastritis and gastric adenocarcinoma. Pathol Res Pract, 2011. 207(10): p. 599-607.
10. Barreto-Zuniga, R., et al., Significance of Helicobacter pylori infection as a risk factor in gastric cancer: serological and histological studies. J Gastroenterol, 1997. 32(3): p. 289-94.
11. Tegtmeyer, N., et al., Helicobacter pylori Employs a Unique Basolateral Type IV Secretion Mechanism for CagA Delivery. Cell Host & Microbe, 2017. 22(4): p. 552-560.e5.
12. Kuipers, E.J., Helicobacter pylori and the risk and management of associated diseases: gastritis, ulcer disease, atrophic gastritis and gastric cancer. Alimentary Pharmacology & Therapeutics, 1997. 11(S1): p. 71-88.
13. Mégraud, F., <em>H pylori</em> antibiotic resistance: prevalence, importance, and advances in testing. Gut, 2004. 53(9): p. 1374.
14. Fiorillo, C., et al., From biology to surgery: One step beyond histology for tailored surgical treatments of gastric cancer. Surgical Oncology, 2020. 34: p. 86-95.
15. Torre, L.A., et al., Global cancer statistics, 2012. CA: a cancer journal for clinicians, 2015. 65(2): p. 87-108.
16. Eaton, K.A., et al., Colonization of gnotobiotic piglets by Helicobacter pylori deficient in two flagellin genes. 1996. 64(7): p. 2445-2448.
17. Eaton, K.A. and S. Krakowka, Effect of gastric pH on urease-dependent colonization of gnotobiotic piglets by Helicobacter pylori. Infection and immunity, 1994. 62(9): p. 3604-3607.
18. Mizote, T., H. Yoshiyama, and T. Nakazawa, Urease-independent chemotactic responses of Helicobacter pylori to urea, urease inhibitors, and sodium bicarbonate. Infection and immunity, 1997. 65(4): p. 1519-1521.
19. Worku, M.L., et al., Motility of Helicobacter pylori in a viscous environment. European journal of gastroenterology & hepatology, 1999. 11(10): p. 1143-1150.
20. Kirschner, D.E. and M.J. Blaser, The dynamics of helicobacter pylori infection of the human stomach. Journal of Theoretical Biology, 1995. 176(2): p. 281-290.
21. Shimizu, T., et al., Helicobacter pylori and the Surface Mucous Gel Layer of the Human Stomach. Helicobacter, 1996. 1(4): p. 207-218.
22. Byrd, J.C., et al., Inhibition of gastric mucin synthesis by Helicobacter pylori. Gastroenterology, 2000. 118(6): p. 1072-1079.
23. Yoshiyama, H. and T. Nakazawa, Unique mechanism of Helicobacter pylori for colonizing the gastric mucus. Microbes and Infection, 2000. 2(1): p. 55-60.
24. Talebi Bezmin Abadi, A., Helicobacter pylori treatment: New perspectives using current experience. Journal of Global Antimicrobial Resistance, 2017. 8: p. 123-130.
25. Eslami, M., et al., Are probiotics useful for therapy of Helicobacter pylori diseases? Comparative Immunology, Microbiology and Infectious Diseases, 2019. 64: p. 99-108.
26. Graham, D.Y., Helicobacter pylori Update: Gastric Cancer, Reliable Therapy, and Possible Benefits. Gastroenterology, 2015. 148(4): p. 719-731.e3.
27. Hu, Y., Y. Zhu, and N.-H. Lu, Novel and effective therapeutic regimens for Helicobacter pylori in an era of increasing antibiotic resistance. Frontiers in cellular and infection microbiology, 2017. 7: p. 168.
28. Huang, A.-H., et al., Impact of Helicobacter pylori antimicrobial resistance on the outcome of 1-week lansoprazole-based triple therapy. Journal of the Formosan Medical Association, 2000. 99(9): p. 704-709.
29. Hsu, P.-I., et al., A prospective randomized trial of esomeprazole-versus pantoprazole-based triple therapy for Helicobacter pylori eradication. American Journal of Gastroenterology, 2005. 100(11): p. 2387-2392.
30. Liou, J.-M., et al., Levofloxacin-based and clarithromycin-based triple therapies as first-line and second-line treatments for Helicobacter pylori infection: a randomised comparative trial with crossover design. Gut, 2010. 59(5): p. 572-578.
31. Wu, L., et al., Effects of anti-H. pylori triple therapy and a probiotic complex on intestinal microbiota in duodenal ulcer. Scientific Reports, 2019. 9(1): p. 12874.
32. Olekhnovich, E.I., et al., Shifts in the Human Gut Microbiota Structure Caused by Quadruple Helicobacter pylori Eradication Therapy. 2019. 10(1902).
33. Cornejo-Pareja, I., et al., H. pylori Eradication Treatment Alters Gut Microbiota and GLP-1 Secretion in Humans. Journal of Clinical Medicine, 2019. 8(4).
34. Imhann, F., et al., Proton pump inhibitors affect the gut microbiome. Gut, 2016. 65(5): p. 740.
35. Jackson, M.A., et al., Proton pump inhibitors alter the composition of the gut microbiota. Gut, 2016. 65(5): p. 749.
36. Pineiro, M. and C. Stanton, Probiotic Bacteria: Legislative Framework— Requirements to Evidence Basis. The Journal of Nutrition, 2007. 137(3): p. 850S-853S.
37. Weizman, Z., G. Asli, and A. Alsheikh, Effect of a probiotic infant formula on infections in child care centers: comparison of two probiotic agents. Pediatrics, 2005. 115(1): p. 5-9.
38. Shornikova, A.-V., et al., Lactobacillus reuteri as a therapeutic agent in acute diarrhea in young children. Journal of pediatric gastroenterology and nutrition, 1997. 24(4): p. 399-404.
39. Lesbros-Pantoflickova, D., I. Corthesy-Theulaz, and A.L. Blum, Helicobacter pylori and probiotics. The Journal of nutrition, 2007. 137(3): p. 812S-818S.
40. Presti, I., et al., Evaluation of the probiotic properties of new Lactobacillus and Bifidobacterium strains and their in vitro effect. Applied Microbiology and Biotechnology, 2015. 99(13): p. 5613-5626.
41. Turková, K., et al., Evaluation of Lactobacillus strains for selected probiotic properties. Folia Microbiologica, 2013. 58(4): p. 261-7.
42. Liévin-Le Moal, V. and A.L. Servin, Anti-infective activities of lactobacillus strains in the human intestinal microbiota: from probiotics to gastrointestinal anti-infectious biotherapeutic agents. Clinical microbiology reviews, 2014. 27(2): p. 167-199.
43. Kim, T.-S., et al., Antagonism of Helicobacter pylori by bacteriocins of lactic acid bacteria. Journal of food protection, 2003. 66(1): p. 3-12.
44. Mukai, T., et al., Inhibition of binding of Helicobacter pylori to the glycolipid receptors by probiotic Lactobacillus reuteri. FEMS Immunology & Medical Microbiology, 2002. 32(2): p. 105-110.
45. Zheng, P.-X., et al., Lactobacillus pentosus strain LPS16 produces lactic acid, inhibiting multidrug-resistant Helicobacter pylori. Journal of Microbiology, Immunology and Infection, 2016. 49(2): p. 168-174.
46. Talarico, T.L., et al., Production and isolation of reuterin, a growth inhibitor produced by Lactobacillus reuteri. Antimicrobial Agents and Chemotherapy, 1988. 32(12): p. 1854.
47. 庞洁, et al., 罗伊氏乳杆菌的益生功能. 中国生物工程杂志, 2011. 31(5): p. 131-137.
48. Valeur, N., et al., Colonization and immunomodulation by Lactobacillus reuteri ATCC 55730 in the human gastrointestinal tract. Applied and Environmental Microbiology, 2004. 70(2): p. 1176-1181.
49. Casas, I.A. and W.J. Dobrogosz, Validation of the Probiotic Concept: Lactobacillus reuteri Confers Broad-spectrum Protection against Disease in Humans and Animals. Microbial Ecology in Health and Disease, 2000. 12(4): p. 247-285.
50. Walter, J., R.A. Britton, and S. Roos, Host-microbial symbiosis in the vertebrate gastrointestinal tract and the Lactobacillus reuteri paradigm. Proceedings of the National Academy of Sciences of the United States of America, 2011. 108 Suppl 1: p. 4645-4652.
51. Singh, T.P., R.K. Malik, and G. Kaur, Cell surface proteins play an important role in probiotic activities of Lactobacillus reuteri. Nutrire, 2016. 41(1): p. 5.
52. Miyoshi, Y., et al., A Mucus Adhesion Promoting Protein, MapA, Mediates the Adhesion of Lactobacillus reuteri to Caco-2 Human Intestinal Epithelial Cells. Bioscience, Biotechnology, and Biochemistry, 2006. 70(7): p. 1622-1628.
53. Roos, S., et al., A collagen binding protein from Lactobacillus reuteri is part of an ABC transporter system? FEMS microbiology letters, 1996. 144(1): p. 33-38.
54. MacKenzie, D.A., et al., Crystal Structure of a Mucus-binding Protein Repeat Reveals an Unexpected Functional Immunoglobulin Binding Activity*. Journal of Biological Chemistry, 2009. 284(47): p. 32444-32453.
55. Walter, J., et al., A high-molecular-mass surface protein (Lsp) and methionine sulfoxide reductase B (MsrB) contribute to the ecological performance of Lactobacillus reuteri in the murine gut. Applied and environmental microbiology, 2005. 71(2): p. 979-986.
56. Mu, Q., V.J. Tavella, and X.M. Luo Role of Lactobacillus reuteri in Human Health and Diseases. Frontiers in microbiology, 2018. 9, 757 DOI: 10.3389/fmicb.2018.00757.
57. Yeo, D., et al., Humulene Inhibits Acute Gastric Mucosal Injury by Enhancing Mucosal Integrity. Antioxidants, 2021. 10(5): p. 761.
58. Knight, J., N. Williams, and Y. Nigam, Gastrointestinal tract 2: the structure and function of the stomach. Nursing Times, 2019. 115(7): p. 43-47.
59. Bellmann, S., et al., Development of an advanced in vitro model of the stomach and its evaluation versus human gastric physiology. Food Research International, 2016. 88: p. 191-198.
60. Feldman, M., H.J. Smith, and T.R. Simon, Gastric emptying of solid radiopaque markers: studies in healthy subjects and diabetic patients. Gastroenterology, 1984. 87(4): p. 895-902.
61. Dressman, J.B., et al., Upper Gastrointestinal (GI) pH in Young, Healthy Men and Women. Pharmaceutical Research, 1990. 7(7): p. 756-761.
62. Tyssandier, V., et al., Processing of vegetable-borne carotenoids in the human stomach and duodenum. American Journal of Physiology-Gastrointestinal and Liver Physiology, 2003. 284(6): p. G913-G923.
63. Thuenemann, E.C., et al., Dynamic Gastric Model (DGM). 2015, Springer, Cham (CH).
64. Minekus, M., The TNO Gastro-Intestinal Model (TIM), in The Impact of Food Bioactives on Health: in vitro and ex vivo models, K. Verhoeckx, et al., Editors. 2015, Springer International Publishing: Cham. p. 37-46.
65. Schneeman, B.O., Gastrointestinal physiology and functions. British Journal of Nutrition, 2002. 88(S2): p. S159-S163.
66. Minekus, M., The TNO gastro-intestinal model (TIM). The impact of food bioactives on health, 2015: p. 37-46.
67. Susan, S., Gray’s anatomy: the anatomical basis of clinical practice. 2015, Elsevier.
68. Liu, W., et al., Development and validation of a new artificial gastric digestive system. Food Research International, 2019. 122: p. 183-190.
69. France, R. and R. Short, Plasma treatment of polymers effects of energy transfer from an argon plasma on the surface chemistry of poly (styrene), low density poly (ethylene), poly (propylene) and poly (ethylene terephthalate). Journal of the Chemical Society, Faraday Transactions, 1997. 93(17): p. 3173-3178.
70. Desmet, T., et al., Nonthermal plasma technology as a versatile strategy for polymeric biomaterials surface modification: a review. Biomacromolecules, 2009. 10(9): p. 2351-2378.
71. Guruvenket, S., et al., Plasma surface modification of polystyrene and polyethylene. Applied surface science, 2004. 236(1-4): p. 278-284.
72. Sharma, A., M. Tomar, and V. Gupta, SnO2 thin film sensor with enhanced response for NO2 gas at lower temperatures. Sensors and Actuators B: Chemical, 2011. 156(2): p. 743-752.
73. Buske, C., Environmentally friendly and cost-saving Atmospheric-pressure plasma technology. ATZproduktion worldwide, 2009. 2(2): p. 18-22.
74. Dong, S., Z. Zhao, and R.H. Dauskardt, Highly transparent bilayer organosilicate multifunctional coatings on plastics deposited by atmospheric plasma deposition with dual organic and inorganic precursors in air. ACS Appl. Mater. Interf, 2015. 7: p. 17929-17934.
75. Pappas, D., Status and potential of atmospheric plasma processing of materials. Journal of Vacuum Science & Technology A: Vacuum, Surfaces, and Films, 2011. 29(2): p. 020801.
76. Jnido, G., G. Ohms, and W. Viöl, Deposition of TiO2 Thin Films on Wood Substrate by an Air Atmospheric Pressure Plasma Jet. Coatings, 2019. 9(7): p. 441.
77. Yvon, M., et al., In vitro simulation of gastric digestion of milk proteins: comparison between in vitro and in vivo data. Journal of agricultural and food chemistry, 1992. 40(2): p. 239-244.
78. Hsu, Y.-T., Evaluation of the inhibitory effect of Tetracycline- Alginate floating beads on the growth of H. pylori in a simulated gastric environment. 2018, National Taiwan university of science and technology. p. 98.
79. Tadjrobehkar, O., Survival and chemotactic behavior of H pylori at different media pH. 2004.
80. Ruoff, K.L., et al., Vancomycin-resistant gram-positive bacteria isolated from human sources. Journal of clinical microbiology, 1988. 26(10): p. 2064.
81. Stevenson, T.H., et al., Growth of Helicobacter pylori in various liquid and plating media. Letters in applied microbiology, 2000. 30(3): p. 192-196.
82. Perez-Perez, G.I., Accurate diagnosis of Helicobacter pylori: Culture, including transport. Gastroenterology Clinics of North America, 2000. 29(4): p. 879-884