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  1. NTU Theses and Dissertations Repository
  2. 醫學院
  3. 生理學科所
Please use this identifier to cite or link to this item: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15867
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???org.dspace.app.webui.jsptag.ItemTag.dcfield???ValueLanguage
dc.contributor.advisor余佳慧
dc.contributor.authorYi-An Shihen
dc.contributor.author施怡安zh_TW
dc.date.accessioned2021-06-07T17:54:04Z-
dc.date.copyright2012-09-19
dc.date.issued2012
dc.date.submitted2012-08-17
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dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15867-
dc.description.abstract對beta-內酰胺有抗藥性的超級細菌,例如擁有新德里金屬beta-內酰胺酶(NDM)-1的超級細菌,對公眾健康有極大的威脅。腸道內的共生菌叢促進腸道屏障功能的穩固,並且與外來病菌競爭。而目前已知道抗生素會擾亂腸道共生菌叢。 目的:了解由抗生素造成的腸道菌叢擾亂,是否會促進超級細菌在腸道內生長和轉移。 方法: BALB/c小鼠給予正常飲水或是抗生素飲水七天,抗生素水去除之後,每隻小鼠灌食109CFU抗氨芐青黴素的大腸桿菌進行感染,在感染後的第0, 1, 3, 7, 和14天進行犧牲。利用蘇木紫-伊紅染色分析腸道的結構,並計數在腸道、脾臟、肝臟的細菌數量。被腸道上皮細胞內吞的細菌數量,則由慶大黴素耐藥檢測分離出來。利用螢光原位雜交,了解細菌入侵到黏膜層的情形。結果: 在第0天,抗生素組的腸道菌叢數量比正常飲水組的低。抗氨芐青黴素的細菌在正常飲水組的腸道中,每個時間點都沒有生長的現象。相反地,抗生素組的小鼠腸道中,在感染後第1, 3天都有抗氨芐青黴素的細菌出現在空腸、盲腸和結腸。抗氨芐青黴素的大腸桿菌在第7和14天被從腸道清除。此外,抗生素組小鼠在感染後第1天,出現盲腸腫脹並且有組織充血及白血球浸潤造成的水腫現象。另外,感染後第三天有細菌入侵空腸腺窩,空腸和結腸也有細菌被腸道上皮細胞內吞的情形。感染後第1和3天,有細菌在脾臟和肝臟轉移的現象。 結論: 正常的腸道菌叢具有屏障功能,可保護腸道不受抗藥性細菌感染。腸道菌叢受到干擾則會促進抗藥性細菌之定殖,並造成腸道共生菌和超級細菌都會體內的散布。zh_TW
dc.description.abstractSuperbugs that are resistant to beta-lactams antibiotics, such as those with New Delhi metallo-beta-lactamase (NDM)-1, pose major threats to public health. Enteric commensal microflora is involved in mucosal barrier fortification and pathogen competition. Antibiotics are known to disrupt intestinal flora. Aim: The aim is to evaluate whether antibiotic-induced intestinal dysbiosis may promote enteric colonization and translocation of superbug. Methods: BALB/c mice were drinking normal water (NW) or antibiotic water (AW) for 7 days. Ampicillin-resistant (Amp-r) E. coli BL21 (109 CFU) was administered by oral gavage after antibiotic withdrawal. Animals were sacrificed at 0, 1, 3, 7 and 14 days after inoculation. The structure of intestine was determined by H&E staining. Bacterial colony forming units in intestine, liver and spleen were assessed. The amount of intracellular bacteria in purified enterocytes was determined using a gentamicin resistance assay. Bacterial invasion to mucosa was determined by fluorescent in situ hybridization. Results: The enteric bacterial counts were reduced in AW mice compared to NW groups on day 0. After inoculation of Amp-r E.coli, no sign of bacterial colonization and translocation was seen in NW mice throughout all time points. In contrast, AW mice showed Amp-r E. coli in the jejunum, cecum and colon after inoculation for 1 and 3 days. Clearance of Amp-r E. coli was associated with recovery of commensal bacterial numbers after 7 and 14 days. Moreover, cecal flatulence and tissue edema associated with hyperemia and leukocyte infiltraton were observed in AW mice on day 1 post-infection. Furthermore, bacterial invasion to jejunal crypts, bacterial endocytosis in jejunal and colonic enterocytes, and bacterial translocation to liver and spleen were observed on day 1 and 3 post-infection in AW mice. Conclusions: The normal commensals served as a barrier to protect the intestine from antibiotic-resistant bacterial colonization. Enteric dysbiosis predisposes antibiotic-resistant bacteria to colonize, leading to systemic dissemination of both commensals and superbug.en
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dc.description.tableofcontents致謝................................................................................................................................I
中文摘要.......................................................................................................................II
英文摘要......................................................................................................................Ⅲ
中英文縮寫名詞對照................................................................................................. .V
一、前言.......................................................................................1
1.腸道組織結構.............................................................................................................1
1.1小腸絨毛結構..................................................................................................1
2.腸道生理功能.............................................................................................................2
2.1物理性屏障......................................................................................................2
2.2化學性屏障......................................................................................................3
2.3免疫性屏障......................................................................................................3
3.腸腔內常生細菌.........................................................................................................4
4.抗生素造成常生菌相失衡.........................................................................................5
5.超級細菌的演進史.....................................................................................................6
6. 實驗目的...................................................................................................................8
二、材料與方法..........................................................................9
1. 實驗動物................................................................................................................9
2. 實驗用細菌............................................................................................................9
2.1含與不含氨芐青黴素的Luria-Bertani培養基瓊脂平板 (LB agar plate) 和培養液 (LB broth) 製備.............................................................................................9
2.1.1 氨芐青黴素儲存液 (Ampicillin stock) 的製備..............................9
2.1.2 LB培養液(LB broth)的製備..............................................................9
2.1.3 LB培養基瓊脂平板(LB agar plate)的製備.......................................10
2.2大腸桿菌生長曲線(growth curve)製作方式.................................................10
2.3餵食小鼠之細菌製備方法............................................................................11
3. 實驗動物分組......................................................................................................11
3.1正常水組 (Normal Water, NW) 飲用水配法..............................................11
3.2抗生素水組(Antibiotic Water, AW)飲用水配法...........................................11
4. 實驗動物感染流程..............................................................................................12
5. 腸道細菌數目變化分析......................................................................................12
5.1腸段總細菌量 (total bacteria) 量分析.........................................................12
5.2細菌轉移數量的分析 (bacteria translocation).............................................12
5.3腸道上皮細胞內吞細菌(endocytosed bacteria in enterocytes)分析........13
6. 組織固定、切片及染色......................................................................................14
6.1石蠟包埋檢體的製備作組織染色(固定液為4%三聚甲醛).................14
6.2石蠟包埋檢體的製備作螢光原位雜交實驗(固定液為Carnoy's Solution)
6.3 冷凍切片包埋檢體的製備作緊密連結螢光染色.......................................15
6.4 蘇木紫-伊紅染色(Haematoxylin and Eosin Staining)............................15
6.5 螢光原位雜交(Fluorescence in situ hybridization, FISH)......................16
6.6緊密連結免疫螢光染色 (Tissue immunofluorescence for ZO-1) .........16
7. 西方轉漬法 (Western blotting)........................................................................17
7.1 黏膜層蛋白質萃取.......................................................................................17
7.2 蛋白質定量...................................................................................................18
7.3蛋白質電泳....................................................................................................18
7.4蛋白質分析....................................................................................................18
8.腸道屏障功能分析................................................................................................20
8.1腸道組織離子通透性(ion permeability)分析..........................................20
8.2腸道組織大分子通透性(macromolecular permeability)分析.................21
三、實驗結果............................................................................22
1. Amp-r E.coli之生長曲線...................................................................................22
2. 抗生素處理與Amp-r E.coli感染對BALB/c小鼠腸道生理的影響................22
2.1小鼠腹腔外觀與消化道外觀變化................................................................22
2.2 腸道組織外觀結構的變化...........................................................................23
3. 腸段總細菌量(total bacteria number)的變化.............................................23
4. 腸道細菌在組織中分布情形..............................................................................24
5. 腸道上皮細胞內吞細菌量(endocytosed bacteria in enterocytes)..............24
6. 細菌轉移至肝臟及脾臟以及血液 (bacteria translocation) 的情形..............25
7. 腸道組織緊密連結ZO-1 和occludin 結構變化.............................................26
8. 小鼠空腸、盲腸及結腸的通透性變化..............................................................26
9. 抗生素水組MAPKs (p38、Erk1/2、JNK) 和I-kappa-B-alpha磷酸化程度的變化......27
四、討論....................................................................................28
五、圖表....................................................................................33
六、參考文獻...........................................................................57
表目錄
表1、腸道共生菌叢的功能.........................................................................................33
表2、正常水組與抗生素水組小鼠在day 0腸道常氧總細菌量的比較...................34
表3、正常水組與抗生素水組小鼠在day 0腸道厭氧總細菌量的比較...................35
圖目錄
圖1、腸道組織結構...................................................................................................36
圖2、腸道腺窩-絨毛軸..............................................................................................37
圖3、Ampicillin resistant E.coli (Amp-r E.coli) 之質體基因圖...........................38
圖4、Amp-r E.coli生長曲線中活菌數量與分光光度計讀數之關係....................39
圖5、正常水組 (Normal water, NW) 和抗生素水組 (antibiotic water, AW)小鼠在灌食Amp-r E.coli之後,腹腔內以及腸道的外觀變化......................................40
圖6-1、空腸組織外觀結構的變化..............................................................................41
圖6-2、盲腸組織外觀結構的變化..............................................................................42
圖6-3、結腸組織外觀結構的變化..............................................................................43
圖7、腸段需氧菌總量(total aerobic bacteria)的變化.......................................44
圖8、腸段厭氧菌總量(total anaerobic bacteria)的變化....................................45
圖9-1、螢光原位雜交分析細菌和E.coli在空腸組織內分佈的情形......................46
圖9-2、螢光原位雜交分析細菌和E.coli在盲腸組織內分佈的情形.....................47
圖9-3、螢光原位雜交分析細菌和E.coli在結腸組織內分佈的情形......................48
圖10、腸道上皮細胞內吞細菌(endocytosed bacteria in enterocytes)變化.........49
圖11、細菌轉移數量的變化 .....................................................................................50
圖12-1、空腸組織緊密連結ZO-1結構變化..............................................................51
圖12-2、盲腸組織緊密連結ZO-1結構變化..............................................................52
圖12-3、結腸組織緊密連結ZO-1結構變化..............................................................53
圖13、抗生素水組腸道黏膜組織中occludin之表現................................................54
圖14、小鼠腸道電生理值和通透性的變化...............................................................55
圖15、抗生素水組小鼠空腸與結腸在MAPKs (p38、ERK、JNK) 和Ikappka B alpha磷酸化程度的變化..............................................................................................................56
dc.language.isozh-TW
dc.subject上皮屏障功能zh_TW
dc.subject抗生素zh_TW
dc.subject超級細菌zh_TW
dc.subject腸道菌叢擾亂zh_TW
dc.subject定殖zh_TW
dc.subject入侵zh_TW
dc.subjectantibioticen
dc.subjectepithelial barrier functionen
dc.subjectinvasionen
dc.subjectcolonizationen
dc.subjectdysbiosisen
dc.subjectsuperbugen
dc.title抗生素引起之共生菌相失衡促使超級細菌在小鼠腸道定殖和入侵zh_TW
dc.titleAntibiotic-induced enteric commensal dysbiosis favours superbug colonization and bacterial invasion in miceen
dc.typeThesis
dc.date.schoolyear100-2
dc.description.degree碩士
dc.contributor.oralexamcommittee張上淳,倪衍玄,盧俊良,賈景山
dc.subject.keyword抗生素,超級細菌,腸道菌叢擾亂,定殖,入侵,上皮屏障功能,zh_TW
dc.subject.keywordantibiotic,superbug,dysbiosis,colonization,invasion,epithelial barrier function,en
dc.relation.page61
dc.rights.note未授權
dc.date.accepted2012-08-17
dc.contributor.author-college醫學院zh_TW
dc.contributor.author-dept生理學研究所zh_TW
Appears in Collections:生理學科所

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