臺灣博碩士論文加值系統

口腔細菌可藉由附著於覆蓋口腔組織表面、由口水蛋白以及從牙齦溝滲出的血清蛋白所形成的薄膜，聚集生長形成牙菌班。在齒齦溝中，齒齦下牙菌斑貼附牙齒表面，不斷刺激牙周組織，促使血液中的嗜中性白血球（Polymorphonuclear neutrophils, PMNs）聚集過來，並穿越上皮連附進入齒齦溝，清除微生物。在感染及感染所造成之輕微發炎反應無法完全去除時，牙菌斑的菌相轉變成以牙周致病菌如Porphyromonas gingivalis（Pg）等菌種為主的菌相，強化免疫反應，進一步造成包括牙周韌帶或齒槽骨等牙周組織的破壞。近年有越來越多研究探討乳酸益生菌對口腔感染之預防或治療效果。這類細菌具有抑制致病菌貼附或入侵細胞、調控發炎因子的分泌、使宿主產生耐受性等特性。雖然有部份研究顯示乳酸益生菌可抑制牙周致病菌的活性，然其是否可藉著影響免疫反應，改變牙周病發生進程，目前仍未知。本研究藉著觀察在以Pg感染大鼠口腔前後不同時間，導入市面常見之乳酸益生菌：Lactobacillus casei strain Shirota（LcS）後，對齒槽骨破壞、全血中免疫細胞、及牙周組織中嗜中性白血球聚集的影響，探討乳酸菌對牙周感染的可能影響。研究結果發現Pg所造成的齒槽骨破壞程度，並未因為在不同時間點給予LcS而改變。全血中的白血球濃度雖然會因為LcS的處理略為增加，但不具統計差異。兩種與牙周發炎有關的白血球：嗜中性白血球與單核球，前者的濃度與其佔白血球的比例未因細菌感染而有明顯變化，後者在有LcS處理的組別有下降的趨勢，然未有統計差異。嗜中性白血球中的活性氧化物質（Reactive oxygen species, ROS），在有給予LcS的組別，Pg感染後先降後升。而只要有細菌處理，無論是LcS、Pg、或是LcS及Pg，無論導入時間先後，牙周上皮中的嗜中性白血球量增加。本研究顯示益生菌或系統性地改變免疫細胞的部份特性，且增加牙周組織中的嗜中性白血球。

Oral bacteria adhere the pellicle, which, on the surface of oral tissues, is formed by proteins in saliva and plasma. Further aggregation and growth of oral bacteria lead to the formation of dental plaque. In the gingival sulcus, the subgingival plaque on the tooth surface stimulates periodontal tissues and recruits polymorphonuclear neutrophils (PMNs). Trans-epithelial migration of PMNs through epithelial attachment is necessary for clearance of microorganisms. When infection and mild inflammation caused by infection can’t be eliminated, the ecology of dental plaque is shifted and dominant by periodontal pathogens such as Porphyromonas gingivalis (Pg). These periodontal pathogens can further strengthen immune responses, leading to a severer destruction of tooth supporting tissues including periodontal ligaments and alveolar bones. Recently, more and more studies are conducted to determine the potential application of probiotics in the prevention and treatment of oral infectious diseases. These bacteria can inhibit the adhesion or invasion of pathogens, regulate proinflammatory factors, or induce host tolerance against microorganisms. As studies have shown that some probiotic strains can inhibit cell viability of periodontal pathogens, its effects on immune responses and related periodontal destruction are still unclear. In this study, Pg infection was established in the rat oral cavities, where the treatment a probiotic strain, Lactobacillus casei strain Shirota (LcS) was also conducted at different time point of Pg infection. Destruction of alveolar bones, immune cells in the blood and presence of PMNs in the periodontal tissues were then observed. Results showed that LcS didn’t change the alveolar bone destruction. White blood cell (WBC) counts were mildly increased by LcS. Concentrations or ratios of cell numbers to WBC counts of PMNs were not changed by LcS. However, LcS reduced the amounts of monocytes. Unfortunately, no statistical difference was observed. After Pg infection, reactive oxygen species in PMNs was first reduced but then increased by LcS. Infection by LcS, Pg, or LcS with Pg increased PMNs in gingiva. This study showed probiotics may change properties of immune cells systemically. They may also stimulate the recruitment of PMNs in periodontal tissues.

目錄 i
圖次目錄 iii
附表目錄 iv
摘要 v
Abstract vii
緒論 1
牙周病的病理機轉 1
Porphyromonas gingivalis 3
嗜中性白血球的防禦機制 4
益生菌乳酸菌 5
研究動機 8
材料與方法 9
細菌培養 9
建立Pg感染造成牙周破壞之大鼠實驗模型 9
益生菌的處理 10
血球分析 10
純化嗜中性白血球 12
測量細胞中ROS 12
大鼠灌流固定（perfusion fixation） 13
蘇木素-伊紅染色（Hematoxylin and eosin stain） 13
組織免疫染色法（Immunohistochemistry） 14
Micro-CT 15
統計分析方法 15
結果 16
先處理LcS再以Pg感染對大鼠免疫細胞及牙周組織之影響 16
1. 對齒槽骨破壞之影響（圖三） 16
2. 對全血中免疫細胞的影響 16
3. 對牙周組織中嗜中性白血球之影響（圖十、圖十一） 18
同時感染LcS和Pg感染對大鼠免疫細胞及牙周組織之影響 19
1. 對齒槽骨破壞之影響（圖十二） 19
2. 對全血中免疫細胞的影響 19
3. 對牙周組織中嗜中性白血球之影響（圖十九、圖二十） 21
先感染Pg再以LcS處理對大鼠免疫細胞及牙周組織之影響 22
1. 對齒槽骨破壞之影響（圖二十一） 22
2. 對全血中免疫細胞的影響 22
3. 對牙周組織中嗜中性白血球之影響（圖二十八、圖二十九） 25
討論 26
圖次目錄
圖 一、建立Pg感染造成牙周破壞之大鼠實驗模型 37
圖 二、感染流程 38
圖 三、預先感染LcS對Pg感染大鼠口腔對齒槽骨之影響 39
圖 四、預先感染LcS對Pg感染大鼠之WBC之影響 40
圖 五、預先感染LcS對Pg感染大鼠之嗜中性白血球之影響 42
圖 六、預先感染LcS對Pg感染大鼠之嗜中性白血球佔WBC比例之影響 44
圖 七、預先感染LcS對Pg感染大鼠之單核球之影響 46
圖 八、預先感染LcS對Pg感染大鼠之單核球佔WBC比例之影響 48
圖 九、預先感染LcS對Pg感染大鼠後ROS變化的影響 50
圖 十、預先感染LcS對Pg感染大鼠後牙周組織的影響 51
圖 十一、預先感染LcS對Pg感染大鼠後牙周組織白血球的影響 52
圖 十二、預先感染LcS對Pg感染大鼠口腔對齒槽骨之影響 54
圖 十三、同時感染LcS對Pg感染大鼠之WBC之影響 55
圖 十四、同時感染LcS對Pg感染大鼠之嗜中性白血球之影響 57
圖 十五、同時感染LcS對Pg感染大鼠之嗜中性白血球佔WBC比例之影響 59
圖 十六、同時感染LcS對Pg感染大鼠之單核球之影響 61
圖 十七、同時感染LcS對Pg感染大鼠之單核球佔WBC比例之影響 63
圖 十八、同時感染LcS對Pg感染大鼠之ROS變化的影響 65
圖 十九、同時感染LcS對Pg感染大鼠後牙周組織的影響 66
圖 二十、同時感染LcS對Pg感染大鼠後牙周組織白血球的影響 67
圖 二十一、後感染LcS對Pg感染大鼠口腔對齒槽骨造成之影響 69
圖 二十二、後感染LcS對Pg感染大鼠之WBC之影響 70
圖 二十三、後感染LcS對Pg感染大鼠之嗜中性白血球之影響 72
圖 二十四、後感染LcS對Pg感染大鼠之嗜中性白血球佔WBC比例之影響 74
圖 二十五、後感染LcS對Pg感染大鼠之單核球之影響 76
圖 二十六、後感染LcS對Pg感染大鼠之單核球佔WBC比例之影響 78
圖 二十七、後感染LcS對Pg感染大鼠ROS變化的影響 80
圖 二十八、後感染LcS對Pg感染大鼠後牙周組織的影響 81
圖 二十九、後感染LcS對Pg感染大鼠後牙周組織白血球的影響 82
附表目錄
附表 一LcS對Pg感染大鼠之紅血球(red blood cells)之影響 84
附表 二 LcS對Pg感染大鼠之血紅素(hemoglobin)之影響 85
附表 三 LcS對Pg感染大鼠之血球容積比(hematocrit)之影響 86
附表 四 LcS對Pg感染大鼠之平均血球容積(mean corpuscular volume)之影響 87
附表 五 LcS對Pg感染大鼠之平均血紅素(mean corpuscular hemoglobin)之影響 88
附表 六 LcS對Pg感染大鼠之平均血色濃度(mean corpuscular hemoglobin concentration)之影響 89
附表 七 LcS對Pg感染大鼠之紅血球分布寬度(red blood cell distribution width (standard deviation))之影響 90
附表 八 LcS對Pg感染大鼠之紅血球分布寬度變異係數(red blood cell distribution width (coefficient variation))之影響 91
附表 九 LcS對Pg感染大鼠之白血球(white blood cells)之影響 92
附表 十 LcS對Pg感染大鼠之嗜中性白血球(neutrophil)之影響 93
附表 十一 LcS對Pg感染大鼠之嗜中性白血球所佔白血球濃度比例之影響 94
附表 十二 LcS對Pg感染大鼠之淋巴球(lymphocyte)之影響 95
附表 十三 LcS對Pg感染大鼠之淋巴球所佔白血球濃度比例之影響 96
附表 十四 LcS對Pg感染大鼠之單核球(monocyte)之影響 97
附表 十五 LcS對Pg感染大鼠之單核球佔白血球比例之影響 98
附表 十六 LcS對Pg感染大鼠之嗜酸性白血球(eosinophil)之影響 99
附表 十七 LcS對Pg感染大鼠之嗜酸性白血球所佔白血球比例之影響 100
附表 十八 LcS對Pg感染大鼠之嗜鹼性白血球(basophil)之影響 101
附表 十九 LcS對Pg感染大鼠之嗜鹼性白血球所佔白血球之影響 102
附表 二十 LcS對Pg感染大鼠之血小板總數(platelets)之影響 103
附表 二十一 LcS對Pg感染大鼠之平均血小板容積(mean platelet volume)之影響 104
附表 二十二 LcS對Pg感染大鼠之血小板容積比(plateletcrit)之影響 105
附表 二十三 LcS對Pg感染大鼠之血小板分布寬度(platelet distribution width)之影響 106
附表 二十四 LcS對Pg感染大鼠之血小板大細胞比率(platelet larger cell ratio)之影響 107
附表 二十五 LcS對Pg感染大鼠之ROS(reactive oxygen species)之影響 108

1. 楊奕馨. 台灣地區成年與老年人口腔健康調查2003-2005. 台灣: 國民健康局; 2006.
2. Cafiero C, Matarasso M, Marenzi G, Iorio Siciliano V, Bellia L, Sammartino G. Periodontal care as a fundamental step for an active and healthy ageing. Scientific World Journal. 2013;2013:127905.
3. Pihlstrom BL, Michalowicz BS, Johnson NW. Periodontal diseases. The Lancet.366:1809-20.
4. Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE. Defining the normal bacterial flora of the oral cavity. J Clin Microbiol. 2005;43:5721-32.
5. Lamont RJ, Jenkinson HF. Life below the gum line: pathogenic mechanisms of Porphyromonas gingivalis. Microbiol Mol Biol Rev. 1998;62:1244-63.
6. Jiao Y, Hasegawa M, Inohara N. Emerging roles of immunostimulatory oral bacteria in periodontitis development. Trends Microbiol. 2014;22:157-63.
7. Freire MO, Van Dyke TE. Natural resolution of inflammation. Periodontol 2000. 2013;63:149-64.
8. Schroeder HE. Transmigration and infiltration of leucocytes in human junctional epithelium. Helv Odontol Acta. 1973;17:6-18.
9. Attstrom R. Presence of leukocytes in crevices of healthy and chronically inflamed gingivae. J Periodontal Res. 1970;5:42-7.
10. Wei S, Kitaura H, Zhou P, Ross FP, Teitelbaum SL. IL-1 mediates TNF-induced osteoclastogenesis. J Clin Invest. 2005;115:282-90.
11. Garlet GP. Destructive and protective roles of cytokines in periodontitis: a re-appraisal from host defense and tissue destruction viewpoints. J Dent Res. 2010;89:1349-63.
12. Darveau RP, Tanner A, Page RC. The microbial challenge in periodontitis. Periodontol 2000. 1997;14:12-32.
13. Hajishengallis G. Immunomicrobial pathogenesis of periodontitis: keystones, pathobionts, and host response. Trends Immunol. 2014;35:3-11.
14. Garrity G. The Bacteroidetes, Spirochaetes, Tenericutes(Mollicutes), Acidobacteria, Fibrobacteres, Fusobacteria, Dictyoglomi, Gemmatimonadetes, Lentisphaerae, Verrucomicrobia, Chlamydiae, and Planctomycetes. In: B. Whitman NRKJTSDRBBPHBJPNLWWLW, editor. Bergey's Manual of Systematic Bacteriology2010.
15. Mysak J, Podzimek S, Sommerova P, Lyuya-Mi Y, Bartova J, Janatova T, et al. Porphyromonas gingivalis: major periodontopathic pathogen overview. J Immunol Res. 2014;2014:476068.
16. Lamont RJ, Chan A, Belton CM, Izutsu KT, Vasel D, Weinberg A. Porphyromonas gingivalis invasion of gingival epithelial cells. Infect Immun. 1995;63:3878-85.
17. Yilmaz O, Watanabe K, Lamont RJ. Involvement of integrins in fimbriae-mediated binding and invasion by Porphyromonas gingivalis. Cell Microbiol. 2002;4:305-14.
18. Lubos E, Mahoney CE, Leopold JA, Zhang YY, Loscalzo J, Handy DE. Glutathione peroxidase-1 modulates lipopolysaccharide-induced adhesion molecule expression in endothelial cells by altering CD14 expression. FASEB J. 2010;24:2525-32.
19. Kato H, Taguchi Y, Tominaga K, Umeda M, Tanaka A. Porphyromonas gingivalis LPS inhibits osteoblastic differentiation and promotes pro-inflammatory cytokine production in human periodontal ligament stem cells. Arch Oral Biol. 2014;59:167-75.
20. Maekawa T, Krauss JL, Abe T, Jotwani R, Triantafilou M, Triantafilou K, et al. Porphyromonas gingivalis manipulates complement and TLR signaling to uncouple bacterial clearance from inflammation and promote dysbiosis. Cell Host Microbe. 2014;15:768-78.
21. Slaney JM, Gallagher A, Aduse-Opoku J, Pell K, Curtis MA. Mechanisms of resistance of Porphyromonas gingivalis to killing by serum complement. Infect Immun. 2006;74:5352-61.
22. Dale BA. Periodontal epithelium: a newly recognized role in health and disease. Periodontol 2000. 2002;30:70-8.
23. Borregaard N. Neutrophils, from marrow to microbes. Immunity. 2010;33:657-70.
24. Mantovani A, Cassatella MA, Costantini C, Jaillon S. Neutrophils in the activation and regulation of innate and adaptive immunity. Nat Rev Immunol. 2011;11:519-31.
25. Waugh DJ, Wilson C. The interleukin-8 pathway in cancer. Clin Cancer Res. 2008;14:6735-41.
26. Scapini P, Lapinet-Vera JA, Gasperini S, Calzetti F, Bazzoni F, Cassatella MA. The neutrophil as a cellular source of chemokines. Immunol Rev. 2000;177:195-203.
27. Kolaczkowska E, Kubes P. Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol. 2013;13:159-75.
28. Nordenfelt P, Tapper H. Phagosome dynamics during phagocytosis by neutrophils. J Leukoc Biol. 2011;90:271-84.
29. Allen LA, Aderem A. Molecular definition of distinct cytoskeletal structures involved in complement- and Fc receptor-mediated phagocytosis in macrophages. J Exp Med. 1996;184:627-37.
30. Segal AW, Dorling J, Coade S. Kinetics of fusion of the cytoplasmic granules with phagocytic vacuoles in human polymorphonuclear leukocytes. Biochemical and morphological studies. J Cell Biol. 1980;85:42-59.
31. Vieira OV, Botelho RJ, Grinstein S. Phagosome maturation: aging gracefully. Biochem J. 2002;366:689-704.
32. Lominadze G, Powell DW, Luerman GC, Link AJ, Ward RA, McLeish KR. Proteomic analysis of human neutrophil granules. Mol Cell Proteomics. 2005;4:1503-21.
33. Mayadas TN, Cullere X, Lowell CA. The multifaceted functions of neutrophils. Annu Rev Pathol. 2014;9:181-218.
34. Vatansever F, de Melo WC, Avci P, Vecchio D, Sadasivam M, Gupta A, et al. Antimicrobial strategies centered around reactive oxygen species--bactericidal antibiotics, photodynamic therapy, and beyond. FEMS Microbiol Rev. 2013;37:955-89.
35. Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, et al. Neutrophil extracellular traps kill bacteria. Science. 2004;303:1532-5.
36. Van Dyke TE, Serhan CN. Resolution of inflammation: a new paradigm for the pathogenesis of periodontal diseases. J Dent Res. 2003;82:82-90.
37. Van Dyke TE. Cellular and molecular susceptibility determinants for periodontitis. Periodontol 2000. 2007;45:10-3.
38. Hajishengallis G, Liang S, Payne MA, Hashim A, Jotwani R, Eskan MA, et al. Low-abundance biofilm species orchestrates inflammatory periodontal disease through the commensal microbiota and complement. Cell Host Microbe. 2011;10:497-506.
39. Guentsch A, Puklo M, Preshaw PM, Glockmann E, Pfister W, Potempa J, et al. Neutrophils in chronic and aggressive periodontitis in interaction with Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. J Periodontal Res. 2009;44:368-77.
40. Jayaprakash K, Demirel I, Khalaf H, Bengtsson T. The role of phagocytosis, oxidative burst and neutrophil extracellular traps in the interaction between neutrophils and the periodontal pathogen Porphyromonas gingivalis. Mol Oral Microbiol. 2015.
41. Chatterjee A, Bhattacharya H, Kandwal A. Probiotics in periodontal health and disease. J Indian Soc Periodontol. 2011;15:23-8.
42. Saxelin M, Tynkkynen S, Mattila-Sandholm T, de Vos WM. Probiotic and other functional microbes: from markets to mechanisms. Curr Opin Biotechnol. 2005;16:204-11.
43. Jonkers D, Penders J, Masclee A, Pierik M. Probiotics in the management of inflammatory bowel disease: a systematic review of intervention studies in adult patients. Drugs. 2012;72:803-23.
44. Parvez S, Malik KA, Ah Kang S, Kim HY. Probiotics and their fermented food products are beneficial for health. J Appl Microbiol. 2006;100:1171-85.
45. Anusha RL, Umar D, Basheer B, Baroudi K. The magic of magic bugs in oral cavity: Probiotics. J Adv Pharm Technol Res. 2015;6:43-7.
46. Caglar E, Kuscu OO, Cildir SK, Kuvvetli SS, Sandalli N. A probiotic lozenge administered medical device and its effect on salivary mutans streptococci and lactobacilli. Int J Paediatr Dent. 2008;18:35-9.
47. Cagetti MG, Mastroberardino S, Milia E, Cocco F, Lingstrom P, Campus G. The use of probiotic strains in caries prevention: a systematic review. Nutrients. 2013;5:2530-50.
48. Krasse P, Carlsson B, Dahl C, Paulsson A, Nilsson A, Sinkiewicz G. Decreased gum bleeding and reduced gingivitis by the probiotic Lactobacillus reuteri. Swed Dent J. 2006;30:55-60.
49. Volozhin AI, Il'in VK, Maksimovskii Iu M, Sidorenko AB, Istranov LP, Tsarev VN, et al. [Development and use of periodontal dressing of collagen and Lactobacillus casei 37 cell suspension in combined treatment of periodontal disease of inflammatory origin (a microbiological study)]. Stomatologiia (Mosk). 2004;83:6-8.
50. Zhu Y, Xiao L, Shen D, Hao Y. Competition between yogurt probiotics and periodontal pathogens in vitro. Acta Odontol Scand. 2010;68:261-8.
51. Zhao JJ, Feng XP, Zhang XL, Le KY. Effect of Porphyromonas gingivalis and Lactobacillus acidophilus on secretion of IL1B, IL6, and IL8 by gingival epithelial cells. Inflammation. 2012;35:1330-7.
52. Michail S, Abernathy F. Lactobacillus plantarum inhibits the intestinal epithelial migration of neutrophils induced by enteropathogenic Escherichia coli. J Pediatr Gastroenterol Nutr. 2003;36:385-91.
53. Carroll IM, Andrus JM, Bruno-Barcena JM, Klaenhammer TR, Hassan HM, Threadgill DS. Anti-inflammatory properties of Lactobacillus gasseri expressing manganese superoxide dismutase using the interleukin 10-deficient mouse model of colitis. Am J Physiol Gastrointest Liver Physiol. 2007;293:G729-38.
54. FIRDI. Almanac of Food Consumption Survey in Taiwan. Taipei: Council of Agriculture, Executive Yuan, R.O.C.; 2008.
55. Kuula H, Salo T, Pirila E, Tuomainen AM, Jauhiainen M, Uitto VJ, et al. Local and systemic responses in matrix metalloproteinase 8-deficient mice during Porphyromonas gingivalis-induced periodontitis. Infect Immun. 2009;77:850-9.
56. de Molon RS, de Avila ED, Boas Nogueira AV, Chaves de Souza JA, Avila-Campos MJ, de Andrade CR, et al. Evaluation of the host response in various models of induced periodontal disease in mice. J Periodontol. 2014;85:465-77.
57. Rogers JE, Li F, Coatney DD, Rossa C, Bronson P, Krieder JM, et al. Actinobacillus actinomycetemcomitans lipopolysaccharide-mediated experimental bone loss model for aggressive periodontitis. J Periodontol. 2007;78:550-8.
58. Lin FY, Hsiao FP, Huang CY, Shih CM, Tsao NW, Tsai CS, et al. Porphyromonas gingivalis GroEL induces osteoclastogenesis of periodontal ligament cells and enhances alveolar bone resorption in rats. PLoS One. 2014;9:e102450.
59. Harikrishnan R, Kim MC, Kim JS, Balasundaram C, Heo MS. Protective effect of herbal and probiotics enriched diet on haematological and immunity status of Oplegnathus fasciatus (Temminck &; Schlegel) against Edwardsiella tarda. Fish Shellfish Immunol. 2011;30:886-93.
60. Chiu YH, Hsieh YJ, Liao KW, Peng KC. Preferential promotion of apoptosis of monocytes by Lactobacillus casei rhamnosus soluble factors. Clin Nutr. 2010;29:131-40.
61. Vong L, Lorentz RJ, Assa A, Glogauer M, Sherman PM. Probiotic Lactobacillus rhamnosus inhibits the formation of neutrophil extracellular traps. J Immunol. 2014;192:1870-7.
62. 顏靖軒. 乾酪乳酸桿菌代田菌對於伴放線桿菌感染口腔鱗狀上皮細胞癌細胞株OECM-1之影響. 台北市: 國立陽明大學; 2013.