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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 余佳慧 | zh_TW |
| dc.contributor.advisor | Linda Chia-Hui Yu | en |
| dc.contributor.author | 林柏諭 | zh_TW |
| dc.contributor.author | Po-Yu Lin | en |
| dc.date.accessioned | 2021-07-11T15:42:25Z | - |
| dc.date.available | 2024-02-28 | - |
| dc.date.copyright | 2018-10-11 | - |
| dc.date.issued | 2018 | - |
| dc.date.submitted | 2002-01-01 | - |
| dc.identifier.citation | 1. Greenwood-Van Meerveld, B., Johnson, A. C., & Grundy, D. (2017). Gastrointestinal physiology and function. In Gastrointestinal Pharmacology (pp. 1-16). Springer, Cham.
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The EMBO journal, 8(2), 621-629. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79081 | - |
| dc.description.abstract | 背景：結腸直腸癌 (colorectal cancer, CRC) 的發生率在台灣已經連續9年佔據十大癌症之首。而發炎性腸道疾病 (inflammatory bowel disease, IBD) 的患者有很高的風險會進展成CRC。先前許多證據指出腸道菌叢與IBD及CRC的病理發展是有關的，但詳細的機制目前尚未清楚。目的：探討含有毒性因子之細菌入侵腸道上皮細胞後是否會直接影響細胞的增生週期，並利用噬菌體抑制細菌之促癌作用。方法：將腸癌模式小鼠大腸上皮細胞分離之大腸桿菌 (Escherichia coli) 與人類大腸癌細胞株Caco-2及T84細胞共培養4小時以測量內化菌落數，並於24小時後利用流式細胞技術觀察細胞週期之改變。利用PCR技術分析內化細菌之毒性因子，並將細菌之fimA、fimH及htrA基因作單獨或共同剔除，隨後與Caco-2細胞共培養後測量內化菌落數及觀察細胞增生情形。噬菌體實驗則是將噬菌體、細菌、Caco-2細胞三者共培養後測量內化細菌菌落數及觀察細胞增生情形。結果：小鼠腸道上皮細胞分離的E. coli感染Caco-2或T84細胞後，都會侵襲至細胞中並促使細胞加速增生。若將細菌毒性因子包括fimA、fimH或htrA基因單獨或共同剔除，則細胞增生情形減少。將針對E. coli有專一性的噬菌體與細菌和Caco-2細胞共培養後，可降低內化菌落數並且減少細胞增生的情形。結論：小鼠腸道上皮細胞分離之細菌透過毒性因子fimA/fimH/htrA內化至細胞中並促進細胞加速增生，推論是細菌促癌的機制。而利用噬菌體可有效抑制細菌促進上皮細胞增生的能力。 | zh_TW |
| dc.description.abstract | Background: The incidence of colorectal cancer (CRC) had been the highest among other types of cancers for 9 years in Taiwan, and patients with inflammatory bowel disease (IBD) had a higher risk. Previous studies showed that gut microbiota were related to IBD and CRC development, but the mechanism is still unclear. Aim: To investigate whether the invasion of intestinal bacteria with virulence factors and endogenous plasmids may promote epithelial cell proliferation, and the inhibitory effect of bacteriophage on bacteria-induced epithelial cell hyperproliferation. Methods: Escherichia coli were isolated from mouse colonocytes of colitis-associated cancer model. Human Caco-2 cells and T84 cells were apically exposed to E. coli for 4 hours to measure the internalized bacterial colony forming units. After 24 hours, the cell cycle progression was measured by flow cytometry. Bacterial virulence genes were determined by PCR; bacteria were deleted of fimA, fimH, and htrA genes individually or simultaneously and then exposed to Caco-2 cells for measuring bacterial internalization and cell proliferation. In addition, Caco-2 cells were exposed to bacteriophage and bacteria for measuring bacterial internalization and cell proliferation. Results: The mouse colonocytic isolated E. coli were internalized into Caco-2 and T84 cells, and caused cell proliferation. The internalized E. coli expressed type 1 fimbriae, fimA, fimH gene or stress protein, htrA gene. Single or multiple gene deletion of fimA, fimH, and htrA in E. coli reduced Caco-2 cell proliferation. Addition of phage in the co-culture of E. coli and Caco-2 cells decreased bacterial internalization and cell proliferation. Conclusions: The mouse colonocytic isolated bacteria invaded epithelial cells and promoted cell proliferation via a fimA/fimH/htrA-dependent mechanism, which may contribute to protumorigenic activity. Bacteriophages inhibited the epithelial hyperproliferation induced by bacterial internalization. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T15:42:25Z (GMT). No. of bitstreams: 1 ntu-107-R05441008-1.pdf: 5115899 bytes, checksum: 5138619f576b1c4a8c4a5df7489a2f38 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 致謝 I
摘要 II Abstract III 目錄 IV 圖表目錄 VII 壹、文獻探討 1 一、腸道屏障功能 (Intestinal barrier function) 1 二、上皮細胞通透性(Epithelial permeability)與細菌位移(Bacterial translocation) 3 2.1 間細胞途徑 (paracellular pathway) 3 2.2 穿細胞途徑 (transcellular pathway) 5 三、腸腔之常生細菌 (Commensal bacteria) 6 3.1 共生菌 (symbionts) 6 3.2 病生菌 (pathobionts) 7 四、細菌之毒性因子 (Bacterial virulence factor) 8 4.1 黏附因子 (adherence factor) 8 4.2 侵入因子 (invasion factor) 9 4.3 免疫反應抑制劑 (immune response inhibitor) 9 4.4 毒素 (toxin) 10 4.5 螯鐵蛋白 (siderophore) 11 4.6 細菌毒性基因水平轉移 (horizontal gene transfer) 11 4.7 具有毒性因子的非病原菌 12 五、細菌內源性質體 (Bacterial endogenous plasmid) 12 5.1 致育性質體 (fertility plasmid, F plasmid) 13 5.2 抗藥性質體 (resistance plasmid, R plasmid) 13 5.3 大腸桿菌素質體 (colicinogenic plasmid, Col plasmid) 13 5.4 降解性質體 (degradative plasmid) 14 5.5 毒性質體 (virulence plasmid) 15 5.6 多功能之質體 15 六、細菌感染之治療方法―抗生素治療 vs. 噬菌體治療 15 6.1 抗生素治療 (antibiotics therapy) 15 6.2 噬菌體治療 (bacteriophage therapy) 16 七、腸道屏障功能失常與腸道菌相失衡之相關疾病 18 7.1 發炎性腸道疾病 (inflammatory bowel disease, IBD) 18 7.1.1 發炎性腸道疾病與腸道屏障功能失常之關聯 19 7.1.2 發炎性腸道疾病與腸道菌相失衡之關聯 19 7.2 結腸直腸癌 (colorectal cancer, CRC) 21 7.2.1 結腸直腸癌與腸道菌叢之關聯 22 八、研究目的 24 貳、材料與方法 25 一、動物模式 25 1.1 發炎性結腸直腸癌模式 (colitis-associated colorectal cancer model) 25 1.2 單獨給予AOM或DSS藥劑組別 28 1.3 結直腸腫瘤數量計數及大小測量 29 二、小鼠腸道上皮細胞之內化細菌及糞便細菌分離 29 2.1 小鼠腸道上皮細胞分離技術 (epithelial cell isolation) 29 2.2 內化細菌之定量分析 (bacteria internalization assay/gentamicin resistant assay) 30 2.3 小鼠糞便均質化 (homogenization) 以及糞便細菌分離 30 三、細菌和毒性因子定性分析 30 3.1 細菌之培養與保存 30 3.2 菌種分析―16S核糖體去氧核醣核酸 (16S ribosomal DNA, 16S rDNA) 定序 31 3.2.1 細菌16S rDNA之聚合酶連鎖反應 (polymerase chain reaction, PCR) 31 3.2.2 PCR產物 (16S rDNA) 純化 31 3.2.3 16S rDNA 定序及分析 32 3.3 細菌毒性因子定量分析 33 3.3.1 細菌毒性因子之聚合酶連鎖反應 (PCR) 33 3.3.2 瓊脂糖膠體電泳 (agarose gel electrophoresis) 34 3.4 細菌毒性因子序列分析 35 3.4.1 萃取細菌之基因體去氧核醣核酸 (genomic DNA) 35 3.4.2 細菌毒性因子之高保真度聚合酶連鎖反應 (High-Fidelity PCR) 36 3.4.3 細菌毒性因子基因定序以及序列比對 (sequence alignment) 37 3.5 細菌生長曲線繪製 37 四、細菌毒性因子基因剔除 37 4.1 單一基因剔除 (single gene deletion) 37 4.2 多重基因剔除 (multiple gene deletion) 39 五、細胞實驗 40 5.1 細菌共培養實驗 41 5.2 細胞中內化細菌計數(bacteria internalization assay/gentamicin resistant assay) 43 5.3 細胞上層培養基之細菌 (apical bacteria) 計數 43 5.4 細菌黏附試驗 (bacterial adhesion assay) 43 六、細胞週期分析 (cell cycle analysis) 和流式細胞術 (flow cytometry) 44 七、噬菌體 (bacteriophage) 實驗 45 7.1 噬菌體增殖與保存 45 7.2 噬菌體點滴試驗 (phage spot assay) 46 7.3 噬菌體溶菌斑試驗 (phage plaque assay) 46 7.4 噬菌體殺菌試驗 (phage killing assay) 47 7.5 噬菌體、細菌與上皮細胞共培養實驗 47 八、內化細菌之內源性質體 (endogenous plasmid) 分析 48 8.1 萃取細菌質體去氧核醣核酸 (plasmid DNA) 48 8.2 限制酶 (restriction enzyme) 截切質體 49 8.3 萃取膠體中去氧核醣核酸 (DNA gel extraction) 49 8.4 建構含內化細菌質體DNA片段之載體 (vector) 50 8.5 轉型作用 (transformation) 51 8.6 內化細菌之質體定序 51 九、統計分析方法 (Statistical analysis) 51 參、結果 53 一、腸道E. coli細菌的內化量與促進上皮細胞增生之能力 53 1.1 比較11種細菌株的內化與促進Caco-2上皮細胞增生能力之差異 53 1.2 內化細菌促進上皮增生的現象亦可見於T84細胞 55 1.3特定毒性因子剔除不會影響細菌在體外環境生長的情形 55 1.4特定毒性因子剔除後會降低細菌促進Caco-2細胞增生的能力 56 二、噬菌體對內化細菌促進上皮細胞增生能力之影響 57 2.1 噬菌體之殺菌效果 57 2.2 內化細菌隨著噬菌體量呈現劑量效應 (dose response) 58 2.3 不同劑量之噬菌體與細菌、細胞共培養後上皮細胞增生情形 59 2.4 不同噬菌體與細菌、細胞共培養後之內化細菌存活量 59 2.5 不同噬菌體與細菌、細胞共培養後上皮細胞增生情形 60 三、腸道分離細菌之毒性因子種類序列和內源性質體分析 60 3.1 小鼠腸道上皮細胞及人類黏膜所分離之大腸桿菌毒性因子種類比對 60 3.2 小鼠腸道上皮細胞及人類黏膜所分離之大腸桿菌毒性因子序列比對 61 3.3 小鼠腸道上皮及人類黏膜之內化細菌有內源性質體 61 3.4 小鼠腸道上皮細胞之內化細菌之內源性質體含有colicin Y基因 62 肆、討論 63 伍、附表與附圖 68 陸、參考文獻 93 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 毒性因子 | zh_TW |
| dc.subject | 內化細菌 | zh_TW |
| dc.subject | 噬菌體治療 | zh_TW |
| dc.subject | 結腸直腸癌 | zh_TW |
| dc.subject | 大腸桿菌 | zh_TW |
| dc.subject | internalized bacteria | en |
| dc.subject | colorectal cancer | en |
| dc.subject | Escherichia coli | en |
| dc.subject | virulence factor | en |
| dc.subject | bacteriophage therapy | en |
| dc.title | 內化細菌毒性和噬菌體對腸道上皮細胞增生與癌化之探討 | zh_TW |
| dc.title | Effects of Internalized Bacterial Virulence and Bacteriophages on Intestinal Epithelial Cell Proliferation and Tumorigenesis | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 106-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 王錦堂;倪衍玄;魏淑? | zh_TW |
| dc.contributor.oralexamcommittee | Jin-Town Wang;Yen-Hsuan Ni;Shu-Chen Wei | en |
| dc.subject.keyword | 結腸直腸癌,內化細菌,大腸桿菌,毒性因子,噬菌體治療, | zh_TW |
| dc.subject.keyword | colorectal cancer,internalized bacteria,Escherichia coli,virulence factor,bacteriophage therapy, | en |
| dc.relation.page | 114 | - |
| dc.identifier.doi | 10.6342/NTU201802825 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2018-08-13 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 生理學研究所 | - |
| dc.date.embargo-lift | 2028-08-10 | - |
| 顯示於系所單位： | 生理學科所 | |
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