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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 陳明汝(Ming-Ju Chen) | |
dc.contributor.author | Yen-Po Chen | en |
dc.contributor.author | 陳彥伯 | zh_TW |
dc.date.accessioned | 2021-06-16T10:14:09Z | - |
dc.date.available | 2018-08-25 | |
dc.date.copyright | 2013-08-25 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-08-19 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60244 | - |
dc.description.abstract | 腸道恆定是維持健康的重要因子。腸道恆定的失恆會因為發炎、病原菌感染等因子所造成,進而導致各種疾病之發生。因此,本論文之目的是以化學法結腸炎與腸道病原菌感染之小鼠動物模式,探討一株自傳統酒精性發酵乳克弗爾粒中所篩選出之菌株Lactobacillus kefiranofaciens M1對維持腸道恆定之影響。本論文亦探討此菌株對於維持腸道恆定之可能機制,包括其可能的受體以及對腸道微生物菌叢之影響。
首先,我們發現Lb. kefiranofaciens M1可在腸道上皮細胞Caco-2單層膜(monolayer)實驗中增進腸道上皮穿膜電阻值(transepithelial electric resistance, TEER)以及腸道上皮修復趨化激素CCL-20之產生。使用聚葡萄糖硫酸酯鈉(dextran sodium sulfate, DSS)造成Caco-2單層膜受損時,Lb. kefiranofaciens M1可減緩DSS引起之TEER值降低以及對巨分子澱粉之穿透性。在動物實驗中,小鼠每日給予108 colony forming unit (CFU)之Lb. kefiranofaciens M1可顯著改善DSS所引起之結腸發炎,並且可減低結腸組織中前發炎細胞激素腫瘤壞死因子-α與介白素-1β之產生,而抗發炎細胞激素介白素-10則顯著提升。我們亦發現類鐸受體(Toll-like receptor, TLR)-2為Lb. kefiranofaciens M1之可能受體,Lb. kefiranofaciens M1可經由TLR2活化核因子-κB(Nuclear factor-κB),且使用TLR2特異性抗體可抑制Lb. kefiranofaciens M1所誘發之CCL-20之產生。在結腸炎動物實驗中,Lb. kefiranofaciens M1無法減緩TLR2基因剔除小鼠因DSS所引起之結腸炎,顯示TLR2的存在與Lb. kefiranofaciens M1保護小鼠抵抗結腸炎有關。 接下來我們探討Lb. kefiranofaciens M1是否可抵抗一種重要之腸內感染菌,出血性大腸桿菌(enterohemorrhagic Escherichia coli, EHEC)之感染。我們發現,小鼠每日給予2 x 108 CFU之Lb. kefiranofaciens M1七日後,可顯著改善EHEC感染所造成之攝食量下降、血便、腸道受損、腎臟受損以及EHEC內臟轉移。此外,血液中之志賀毒素(Shiga-toxin, Stx)亦顯著減少。此抗感染功能可能是因為Lb. kefiranofaciens M1可顯著提升黏膜特異性抗EHEC免疫球蛋白A(mucosal specific anti-EHEC immunoglobulin A)之產生。抗感染之機制亦使用Caco-2腸道上皮細胞來探討,我們發現Lb. kefiranofaciens M1可減少EHEC引起之腸道上皮死亡以及腸道上皮單層膜之傷害。但是Lb. kefiranofaciens M1並無法直接中和Stx及其引起之毒性。 然而,我們並不知道Lb. kefiranofaciens M1調節腸道之功能是否經由直接引起動物體之反應,或是經由改變腸道菌叢(intestinal microbiota)而引起。因此我們使用無菌小鼠(germ-free mice)來探討Lb. kefiranofaciens M1在沒有腸道菌叢之存在下,直接對小鼠之影響。我們發現口服給予Lb. kefiranofaciens M1亦可以在無菌小鼠中改善DSS所引起之結腸發炎,其腸道出血評分、結腸縮短及結腸組織切片評估中,皆有顯著之改善。此外,Lb. kefiranofaciens M1可增加迴腸絨毛-腺窩軸(villus-crypt axis)之長度以及杯狀細胞之數量。在免疫調節功能中,口服給予Lb. kefiranofaciens M1可增加脾臟細胞經TLR活化後所產生之Th1細胞激素干擾素-γ與介白素-12。以上可證明Lb. kefiranofaciens M1可在沒有腸道菌叢之存在下直接調節腸道功能與免疫調節。然而,Lb. kefiranofaciens M1卻無法定殖於腸道。 Lb. kefiranofaciens M1對於腸道功能之非直接影響則可由分析腸道菌叢菌相而得知,我們使用次世代焦磷酸定序法(pyrosequencing)針對腸內細菌之16S rDNA進行定序分析。小鼠於口服Lb. kefiranofaciens M1後可增加腸道菌叢中Firmicutes/Bacteroidetes之比例與Lactobacillus,但會減少Barnesiella。而TLR2基因剔除小鼠在口服Lb. kefiranofaciens M1後會增加較多之Firmicutes。這部分證明了Lb. kefiranofaciens可改變腸道菌相,而可能為其維持腸道恆定之機制之一。 本論文之成果證明Lb. kefiranofaciens M1具有維持腸道恆定功能之益生菌特性。我們期望Lb. kefiranofaciens M1可發展為一種新的可應用於人類或是動物的調節腸道恆定益生菌產品。 | zh_TW |
dc.description.abstract | Intestinal homeostasis is one of the important parameters to human health. The imbalance of intestinal homeostasis caused by inflammation, pathogen and other factors might further trigger other diseases. The purpose of this study was to investigate the effects of Lactobacillus kefiranofaciens M1, which was a potential probiotic strain isolated from grains of a fermented milk beverage kefir previously, on the regulation of intestinal homeostasis through cellular and animal models. The possible underlying mechanisms including putative receptors, host-microbial interactions and alteration of intestinal microbiota were investigated as well.
At first we found that Lb. kefiranofaciens M1 could reduce chemical-induced loss of epithelial barrier in vitro. Treatment of Lb. kefiranofaciens M1 enhanced transepithelial electric resistance (TEER) value and intestinal restitution chemokine CCL-20 production in intestinal epithelial monolayer constructed by Caco-2 cell line. Further, pre-treatment of Lb. kefiranofaciens M1 protected Caco-2 intestinal epithelial monolayer against dextran sodium sulfate (DSS)-induced loss of barrier function by TEER and macromolecule permeability assay. The in vivo effects were proved that Lb. kefiranofaciens M1 could ameliorate DSS-induced colitis with a significant attenuation of the bleeding score and colon length shortening in mice. Production of pro-inflammatory cytokines was decreased and the anti-inflammatory cytokine IL-10 was increased in intestinal tissues of the DSS-treated mice given Lb. kefiranofaciens M1. The putative receptor for the protective effects of Lb. kefiranofaciens M1 against DSS-induced damage was Toll-like receptor (TLR)-2, which was involved in Lb. kefiranofaciens M1-induced cytokine production in vitro and in attenuation of the bleeding score and colon length shortening in vivo. Next we investigated the effects of Lb. kefiranofaciens M1 on a foodborne pathogen, enterohemorrhagic Escherichia coli (EHEC), which could disrupt intestinal epithelial function and cause severe hemolytic colitis. We found that oral administration of Lb. kefiranofaciens M1 was able to prevent EHEC infection-induced symptoms, intestinal damage, renal damage, bacteria translocation and Shiga toxin penetration in mice. Furthermore, the mucosal EHEC specific immunoglobulin-A response were increased after Lb. kefiranofaciens M1 administration in EHEC infected mouse system. Additionally, in vitro, Lb. kefiranofaciens M1 was shown to have a protective effect on Caco-2 intestinal epithelial cells and Caco-2 intestinal epithelial cell monolayers, where Lb. kefiranofaciens M1 limited EHEC-induced cell death and reduced the loss of epithelial integrity respectively. However, whether the effects of Lb. kefiranofaciens M1 elicited was acted directly to host or through regulating host’s intestinal microbiota was unknown. Thus, we investigated the effects of Lb. kefiranofaciens M1 on germ-free (GF) mice, which was lack of microbiota throughout, to determine the direct effects of the potential probiotic on host itself. We also investigated whether administration with Lb. kefiranofaciens M1 could alter intestinal microbiota community of conventional mice by next-generation sequencing technology targeting to bacterial 16S rDNA. Results showed that oral inoculation of Lb. kefiranofaciens M1 could ameliorate symptoms in DSS-induced colitis in GF mice. Length of villus-crypt axis and number of goblet cells of ileum were also increased. Th1 cytokine responses upon TLR stimulation were enhanced in GF mice inoculated with Lb. kefiranofaciens M1. However, Lb. kefiranofaciens M1 failed to colonize in host. These evidences proved that Lb. kefiranofaciens M1 could conduct the beneficial effects on host without regulation of microbiota. The effects of administration of Lb. kefiranofaciens M1 on intestinal microbiota composition were investigated by using next-generation pyrosequencing technique targeting to 16S rDNA amplicon on bacterial genome. The results showed that administration of Lb. kefiranofaciens M1 could increase Firmicutes to Bacteroidetes ratio and population of Lactobacillus spp., but decrease Barnesiella spp. in intestinal microbiota. On the other hand, TLR2 might participate in the shaping effect of Lb. kefiranofaciens M1 on intestinal microbiota, but in an unknown way. These findings supported that alteration of intestinal microbiota composition might be a possible mechanisms of Lb. kefiranofaciens M1 eliciting probiotic ability. The findings of this study prove the potential probiotic activity of Lb. kefiranofaciens M1 in sustaining intestinal homeostasis in many aspects. Lactobacillus kefiranofaciens M1 could be applied as a novel potential probiotic to improve intestinal health in both human and animals in the future. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T10:14:09Z (GMT). No. of bitstreams: 1 ntu-102-D96626003-1.pdf: 9534955 bytes, checksum: be57fa3e29d5200f1466851a17141dd1 (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | Abstract in Chinese i
Abstract in English iii Introduction 1 Chapter 1: Literature review 6 1-1 Intestinal barrier function 6 1-1-1 barrier components 10 1-1-1-1 Mucus layer 10 1-1-1-2 Intestinal epithelial cell 12 1-1-1-3 Toll-like receptors 14 1-1-2 Intestinal barrier functions vs. cellular mechanisms 18 1-1-2-1 Nuclear factor-κB 18 1-1-2-2 Peroxisome proliferating-activated receptor-γ 19 1-1-3 Intestinal barrier functions vs. innate immunity 20 1-1-3-1 Antimicrobial peptides 20 1-1-3-2 Lysozyme C 20 1-1-3-3 Phospholipase 21 1-1-3-4 C-type lectin 21 1-1-4 Intestinal barrier functions vs. mucosal IgA 21 1-2 Effects of commensal bacteria and probiotics on intestinal barrier function 24 1-2-1 Commensal bacteria vs. intestinal barrier function 24 1-2-2 Probiotics vs. intestinal barrier function 26 1-2-2-1 Mucus layer 26 1-2-2-2 Tight junction 28 1-2-2-3 Antimicrobial peptides 28 1-2-2-4 Mucosal IgA 29 1-3 Studying intestinal barrier function 30 1-3-1 Cellular models 30 1-3-2 Chemical-induced colitis mouse models 32 1-3-2-1 Dextran sodium sulfate model 32 1-3-2-2 Effects of probiotics on intestinal function using DSS-induced colitis model 33 1-3-2-2-1 Gram-positive probiotic bacteria 33 1-3-2-2-2 Gram-negative probiotic bacteria 35 1-3-2-2-3 Probiotic yeasts 36 1-3-2-2-4 Mixed probiotic bacteria 36 1-3-3 Enterohemorrhagic Escherichia coli induced colitis 37 1-3-3-1 Enterohemorrhagic Escherichia coli 37 1-3-3-2 Effects of probiotics on intestinal function using enterohemorrhagic Escherichia coli model 38 1-3-3-2-1 In vitro studies 39 1-3-3-2-2 In vivo studies 41 1-4 Intestinal microbiota 42 1-4-1 Development of intestinal microbiota 42 1-4-2 Intestinal microbiota and health 44 1-4-2-1 Effects on metabolism 44 1-4-2-2 Effects on immunoregulation 44 1-4-2-3 Effects on intestinal function and morphology 46 1-4-2-4 Effects on bone homeostasis 46 1-4-2-5 Effects on behavior 46 1-4-2-6 Delineation of intestinal microbiota composition in health 46 1-4-3 Intestinal microbiota and disease 47 1-4-3-1 Effects of inflammatory bowel disease 48 1-4-3-2 Effects of colorectal cancer 48 1-4-3-3 Effects of allergies 48 1-4-3-4 Effects of diabetes 49 1-4-3-5 Effects of obesity 49 1-4-3-6 Effects of anorexia 49 1-4-3-7 Effects of autism 50 1-4-4 Intestinal microbiota and probioics 50 1-5 Methods of studying effects of intestinal microbiota 52 1-5-1 Next generation pyrosequencing 52 1-5-1-1 Theory and equipment 52 1-5-1-2 Application of next generation pyrosequencing on probiotic analysis 56 1-5-2 Germ-free and gnotobiotic mice model 61 1-5-2-1 Production of germ-free mice 61 1-5-2-2 Intestinal characteristics of germ-free mice 63 1-5-2-3 Application of germ-free mice to probiotic research 64 1-6 Kefir 66 1-6-1 General aspects 66 1-6-2 Microbial composition 66 1-6-3 Formation of kefir grain 69 1-6-4 Functional properties 71 1-6-5 Lactobacillus kefiranofaciens 73 Chapter 2: Investigation of the effect of Lactobacillus kefiranofaciens M1 on the intestinal homeostasis through chemical–induced models 74 2-1 Objectives 74 2-2 Experimental design 74 2-3 Material and methods 75 2-3-1 Lactic acid bacterial sample preparation 75 2-3-2 Caco-2 epithelial monolayer 75 2-3-3 Effect of Lb. kefiranofaciens M1 on integrity of Caco-2 epithelial monolayer 76 2-3-4 DSS-induced colitis animal model 76 2-3-5 Intestinal bleeding assessment 77 2-3-6 Histological evaluation 77 2-3-7 Colon organ culture 80 2-3-8 Splenocyte isolation 80 2-3-10 Flow cytometry for splenocyte and Peyer’s patch cells 81 2-3-12 Reporter gene assay 81 2-3-11 Antibody blocking test 82 2-3-12 Statistical analysis 82 2-4 Results 83 2-4-1 Lactobacillus kefiranofaciens M1 enhances intestinal epithelial barrier function in vitro 83 2-4-2 Lactobacillus kefiranofaciens M1 protects intestinal epithelial monolayer against DSS challenge in vitro 83 2-4-3 Lactobacillus kefiranofaciens M1 ameliorates DSS-induced colitis in vivo 86 2-4-4 Effects of administration of Lb. kefiranofaciens M1 on cytokines secretion in colonic organ culture 90 2-4-5 Administration of Lb. kefiranofaciens M1 does not change splenic and PP’s CD4+CD25+ cell population 90 2-4-6 Heat-killed Lb. kefiranofaciens M1 is not able to ameliorate DSS-induced colitis 90 2-4-7 Effect of Lb. kefiranofaciens M1 on healing model of DSS-induced colitis 96 2-4-8 TLR2 plays an important role through NFκB in protective effects of Lb. kefiranofaciens M1 on DSS-induced colitis 100 2-5 Discussion 103 2-6 Conclusion 109 Chapter 3: Investigation of the effect of Lactobacillus kefiranofaciens M1 on the intestinal homeostasis through pathogenic-induced animal models 110 3-1 Objectives 110 3-2 Experimental design 110 3-3 Material and Methods 111 3-3-1 Lactobacillus kefiranofaciens M1 sample preparation 111 3-3-2 Enterohemorrhagic E. coli O157:H7 preparation 111 3-3-3 Enterohemorrhagic E. coli infection model 111 3-3-4 Fecal bleeding assessment 113 3-3-5 Analysis of EHEC O157:H7 amount in organs and blood 113 3-3-6 Histological evaluation 114 3-3-7 Measurement of immunoglobulin production 114 3-3-8 Measurement of Stx production 115 3-3-9 Assessment of intestinal epithelial cell viability 115 3-3-10 Caco-2 intestinal epithelial cell monolayer preparation 116 3-3-11 Measurement of Caco-2 cell monolayer integrity 117 3-3-12 Statistical analysis 117 3-4 Results 118 3-4-1 Administration of Lb. kefiranofaciens M1 ameliorates the symptoms of EHEC infection 118 3-4-2 Administration of Lb. kefiranofaciens M1 prevents intestinal architecture damage and reduces renal injury induced by EHEC infection 118 3-4-3 Administration of Lb. kefiranofaciens M1 decreases EHEC translocation and the Stx-1/Stx-2 level in serum 122 3-4-4 Administration of Lb. kefiranofaciens M1 increases mucosal EHEC-specific immunoglobulin response 128 3-4-5 Putative mechanisms by which Lb. kefiranofaciens M1 can protect against EHEC in vitro 128 3-5 Discussion 133 3-6 Conclusion 109 Chapter 4: Investigation of the effects of Lactobacillus kefiranofaciens M1 on germ-free mice 137 4-1 Objectives 137 4-2 Experimental design 137 4-3 Material and methods 138 4-3-1 Lactobacillus kefiranofaciens M1 preparation 138 4-3-2 Gnotobiotic mice generation with Lb. kefiranofaciens M1 138 4-3-3 Fecal lactic acid bacteria count 140 4-3-4 Goblet cell count 140 4-3-5 Splenocyte isolation 140 4-3-6 Toll-like receptor agonist treatment 141 4-3-7 Cytokine production 141 4-3-8 Dextran sodium sulfate-induced colitis 141 4-3-9 Statistical analysis 142 4-4 Results 143 4-4-1 Lactobacillus kefiranofaciens M1 ameliorates cecal enlargement and stimulate intestinal epithelial cell growth in GF mice 143 4-4-2 Lactobacillus kefiranofaciens M1 ameliorates DSS-induced colitis in GF mice 143 4-4-3 Lactobacillus kefiranofaciens M1 enhances TLR agonist-induced Th1 cytokine responses in splenocyte 147 4-4-4 Lactobacillus kefiranofaciens M1 could not colonize in GF mice 151 4-5 Discussion 153 4-6 Conclusion 156 Chapter 5: Investigation of the effects of Lactobacillus kefiranofaciens M1 on intestinal microbiota composition by using pyrosequencing 157 5-1 Objectives 157 5-2 Experimental design 157 5-3 Material and methods 158 5-3-1 Animal experiment group 158 5-3-2 Fecal sample collection 158 5-3-3 Fecal DNA extraction 159 5-3-4 Primer design 159 5-3-5 Polymerase chain reaction for 16S rDNA amplicons 160 5-3-6 Pyrosequencing 160 5-4 Results 162 5-4-1 Run summary 162 5-4-2 Effect of administration of live or heat-inactivated Lb. kefiranofaciens M1 on intestinal microbiota composition in mice 162 5-4-3 Effects of administration of Lb. kefiranofaciens M1 on intestinal microbioata composition in wild type and TLR2 knock-out mice 170 5-5 Discussion 170 5-6 Conclusion 174 Chapter 6: Conclusion 175 References 177 Author’s biography 214 | |
dc.language.iso | en | |
dc.title | 克弗爾分離菌株Lactobacillus kefiranofaciens M1對腸道功能調節之影響與機制探討 | zh_TW |
dc.title | Effects and mechanisms of Lactobacillus kefiranofaciens M1 on regulation of intestinal function | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 林慶文(Chin-Wen Lin),周正俊(Cheng-Chun Chou),曾浩洋(Hau-Yang Tsen),蔡英傑(Ying-Chieh Tsai),潘子明(Tsu-Ming Pan) | |
dc.subject.keyword | 克弗爾,Lactobacllus kefiranofaciens M1,益生菌,腸道保健, | zh_TW |
dc.subject.keyword | kefir,Lactobacillus kefiranofaciens M1,probiotic,intestinal health, | en |
dc.relation.page | 214 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2013-08-19 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 動物科學技術學研究所 | zh_TW |
顯示於系所單位: | 動物科學技術學系 |
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