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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 | 王錦堂(Jin-Town Wang) | |
dc.contributor.author | Fang-Chi Chang | en |
dc.contributor.author | 張芳齊 | zh_TW |
dc.date.accessioned | 2021-07-09T15:53:46Z | - |
dc.date.available | 2022-08-28 | |
dc.date.copyright | 2019-08-28 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-07-22 | |
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Koeth RA, Wang Z, Levison BS, Buffa JA, Org E, Sheehy BT, et al. Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. Nature medicine. 2013;19(5):576-85. 24. Rebouche CJ. Carnitine function and requirements during the life cycle. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 1992;6(15):3379-86. 25. Rebouche CJ, and Chenard CA. Metabolic fate of dietary carnitine in human adults: identification and quantification of urinary and fecal metabolites. The Journal of nutrition. 1991;121(4):539-46. 26. Zhu Y, Jameson E, Crosatti M, Schafer H, Rajakumar K, Bugg TD, et al. Carnitine metabolism to trimethylamine by an unusual Rieske-type oxygenase from human microbiota. Proceedings of the National Academy of Sciences of the United States of America. 2014;111(11):4268-73. 27. Bennett BJ, de Aguiar Vallim TQ, Wang Z, Shih DM, Meng Y, Gregory J, et al. Trimethylamine-N-oxide, a metabolite associated with atherosclerosis, exhibits complex genetic and dietary regulation. Cell metabolism. 2013;17(1):49-60. 28. Treacy EP, Akerman BR, Chow LM, Youil R, Bibeau C, Lin J, et al. Mutations of the flavin-containing monooxygenase gene (FMO3) cause trimethylaminuria, a defect in detoxication. Human molecular genetics. 1998;7(5):839-45. 29. Wang Z, Klipfell E, Bennett BJ, Koeth R, Levison BS, Dugar B, et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature. 2011;472(7341):57-63. 30. Bremer J. Carnitine--metabolism and functions. Physiological reviews. 1983;63(4):1420-80. 31. Neville SA, Lecordier A, Ziochos H, Chater MJ, Gosbell IB, Maley MW, et al. Utility of matrix-assisted laser desorption ionization-time of flight mass spectrometry following introduction for routine laboratory bacterial identification. Journal of clinical microbiology. 2011;49(8):2980-4. 32. van Veen SQ, Claas EC, and Kuijper EJ. High-throughput identification of bacteria and yeast by matrix-assisted laser desorption ionization-time of flight mass spectrometry in conventional medical microbiology laboratories. Journal of clinical microbiology. 2010;48(3):900-7. 33. Wang WL, Xu SY, Ren ZG, Tao L, Jiang JW, and Zheng SS. Application of metagenomics in the human gut microbiome. World journal of gastroenterology. 2015;21(3):803-14. 34. Tang WH, Wang Z, Levison BS, Koeth RA, Britt EB, Fu X, et al. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. The New England journal of medicine. 2013;368(17):1575-84. 35. Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M, et al. Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association. Circulation. 2016;133(4):e38-360. 36. Wu WK, Chen CC, Liu PY, Panyod S, Liao BY, Chen PC, et al. Identification of TMAO-producer phenotype and host-diet-gut dysbiosis by carnitine challenge test in human and germ-free mice. Gut. 2019;68(8):1439-49. 37. Goodman AL, Kallstrom G, Faith JJ, Reyes A, Moore A, Dantas G, et al. Extensive personal human gut microbiota culture collections characterized and manipulated in gnotobiotic mice. Proceedings of the National Academy of Sciences of the United States of America. 2011;108(15):6252-7. 38. Henriques HN, de Carvalho AC, Soares Filho PJ, Pantaleao JA, and Guzman-Silva MA. Effect of prolonged use of high dose of tibolone on the vagina of ovariectomized rats. International journal of experimental pathology. 2011;92(4):266-71. 39. Goodman AL, Kallstrom G, Faith JJ, Reyes A, Moore A, Dantas G, et al. Extensive personal human gut microbiota culture collections characterized and manipulated in gnotobiotic mice. Proceedings of the National Academy of Sciences. 2011;108(15):6252-7. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76525 | - |
dc.description.abstract | 近期的研究發現腸道微生物群可能在冠狀動脈疾病中扮演重要角色,傳統以糞便檢體為研究對象並以16S rDNA 定序為普遍鑑別菌種的方法,但現在培養體系統日趨重要,我們經實驗發現高登培養基 (Gordon’s medium) 與五盤日本伊藤非選擇性培養基 (Itoh’s non-selective medium) 對比於十一盤選擇性培養基 (Itoh’s selective medium) 可能培養出最多菌種,因此我們將主要使用此進行後續實驗。關於糞便檢體收集後的儲存方式,實驗發現運輸培養液 (Puritan’s transportation medium)雖然維持了較多新鮮檢體的操作分類單元 (operational taxonomic unit, OTU) 但是不穩定,而冷凍檢體只比新鮮檢體少約30%~50%的OTU。且經實驗證實基質輔助雷射脫附游離飛行時間質譜儀目前鑑定之圖譜訊號弱、資料庫少且鑑定效率約只有16S rDNA 定序的40%。而與我們合作的實驗室利用次世代定序 (next generation sequencing) 發現食用左旋肉鹼 (L-carnitine) 後血漿產生低量氧化三甲胺(trimethylamine-N-oxide, TMAO)的受試者糞便中出現頻率高且量顯著多的腸內菌為Flavonifractor plautii (F.plautii),推測其可能不利於氧化三甲胺的產生。並且我以相同的培養系統加以專一性的聚合酶連鎖反應 (polymerase chain reaction) 分離出了F.plautii。我們首先想探討是否其會分解三甲胺從而達到抑制氧化三甲胺的生成,實驗結果顯示並不會分解,其次我們也想知道是否其會與高量氧化三甲胺生成之菌反應,而抑制三甲胺的分泌,在膽鹼與肉鹼之代謝利用實驗中發現其並不會抑制。我們同樣也想知道是否其會抑制高量氧化三甲胺生成之菌生長,但在競爭實驗中並無發現抑制現象。
因此得出結論:高登培養基與日本伊藤非選擇性培養基為較適宜的腸內菌培養基,能培養出最多菌種;冷凍糞便檢體OTU數目只比新鮮檢體少約30%~50%的OTU,因此篩選菌種時先使用冷凍檢體,而有特殊菌種培養困難時再採用新鮮糞便檢體;16S rDNA 定序為較好的鑑定菌種方式;F.plautii並不會分解三甲胺;也不會抑制高量氧化三甲胺生成之菌的三甲胺分泌;同樣並未發現能抑制高量氧化三甲胺生成之菌的生長。其功能有待進一步研究。 | zh_TW |
dc.description.abstract | Recent studies have also suggested that gut microbiota could be an important role in the development of CAD. Although stool sample is traditionally used for 16S rDNA sequencing to identify bacterial species and is considered as a common and convenient way to identify bacterial species, culturomic system is increasingly important nowadays. Based on our scientific experiments, Gordon’s medium and five Itoh’s non-selective medium can culture the most bacteria, so we chose them to continue on our studies. While Puritan’s transportation medium can maintain a great amount of operational taxonomic unit (OTU) of fresh stool sample, it is not stable, and frozen sample is only 30%~50% less OTU than fresh sample. In our experiments, we found out that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) detecting signals for our bacteria are weak and lacking of database, the identification efficiency is 60% less that 16S rDNA sequencing. And our collaborative laboratory found out that healthy subjects challenged with L-carnitine who produced low trimethylamine-N-oxide (TMAO) had a high frequency of F.plautii, suggesting that this species is disadvantageous to the production of TMAO. In my experiment, I use the same culturomic system with specific polymerase chain reaction and successfully isolate F.plautii. First we want to know if F.plautii can degrade TMA in order to inhibit the production of TMAO, experiment showed that it cannot degrade TMA, we also want to know if it will interact with high TMAO producing bacteria to inhibit the producing of TMAO. But in the L-carnitine/Choline assay, we found out that it does not have the property of inhibition. We also like to know if F.plautii can inhibit the growth of high TMAO producing bacteria, but did not find it in the competing assay.
My conclusions are Gordon’s medium and five Itoh’s non-selective medium are better for gut microbiota, for they can culture the most bacteria; frozen sample is only 30%~50% less OTU than fresh sample, so we decide to use frozen sample for screening bacteria, and collect fresh sample only when having difficulty in culturing some bacterial species; 16S rDNA sequencing is a better way to identify bacterial species; F.plautii cannot degrade TMA nor inhibit the formation of TMAO of high TMAO producing bacteria or their growth. Its function needs further study. | en |
dc.description.provenance | Made available in DSpace on 2021-07-09T15:53:46Z (GMT). No. of bitstreams: 1 ntu-108-R06445107-1.pdf: 1898359 bytes, checksum: 8b36ca4b173eb3ad0255df119f35ab1d (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 口試委員審定書 ii
致謝 iii 中文摘要 iv Abstract vi 目 錄 viii 圖目錄 xi 表目錄 xii 第一章、 緒論 - 1 - 1.1腸道微生物群(Gut microbiota) - 1 - 1.2腸道微生物群與宿主的免疫系統 - 1 - 1.3冠狀動脈疾病與腸道微生物群的關聯 - 2 - 1.4基質輔助雷射脫附游離飛行時間質譜儀 (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF MS) - 4 - 1.5次世代定序(Next Generation Sequencing , NGS)之微生物體16S擴增子定序 (16S amplicon sequencing) - 5 - 1.6高效液相層析儀 (high performance liquid chromatography, HPLC) - 5 - 1.7研究動機 - 6 - 第二章、 材料與方法 - 7 - 2.1 材料 - 7 - 2.1.1培養基 (media) - 7 - 2.1.2引子 (primers) - 8 - 2.2 方法 - 8 - 2.2.1 糞便之腸道微生物培養 (Culturing of fecal microbiota) - 8 - 2.2.2 萃取細菌之DNA (Genomic DNA extraction) - 8 - 2.2.3 聚合酶連鎖反應 (polymerase chain reaction, PCR) - 9 - 2.2.4 基質輔助雷射脫附游離飛行時間質譜儀 (MALDI-TOF)樣品前處理 - 10 - 2.2.5 序列稀釋分離菌種 - 11 - 2.2.6 以特定序列分離菌種 - 11 - 2.2.7 三甲胺分解實驗 (Trimethylamine assay, Tma assay) - 12 - 2.2.8 膽鹼與肉鹼之代謝利用實驗 (L-carnitine/Choline assay) - 12 - 2.2.9 氘標記膽鹼與肉鹼之代謝利用實驗 (d9-L-carnitine / d9-Choline assay) - 12 - 2.2.10 勝任細胞 (competent cell) 的製備 - 13 - 2.2.11 電穿孔 (electroporation) - 13 - 2.2.12 競爭實驗 (competing assay) - 13 - 第三章、 結果 - 14 - 3.1 探討不同培養基對菌種數目的影響 - 14 - 3.2 以不同儲存方式與時間對糞便檢體菌種數目的影響 - 14 - 3.3 以基質輔助雷射脫附游離飛行時間質譜儀進行菌種鑑定 - 15 - 3.4 高登培養基 (Gordon’s medium, GM) 培養之菌株以16S rDNA定序進行菌種鑑定 - 15 - 3.5 日本伊藤選擇性培養基培養之菌株以16S rDNA定序進行菌種鑑定 - 16 - 3.6 以聚合酶連鎖反應 (polymerase chain reaction, PCR) 分離菌種 - 16 - 3.7 以特定序列分離菌種 - 17 - 3.8 三甲胺分解實驗 (Trimethylamine assay, Tma assay) - 17 - 3.9膽鹼與肉鹼之代謝利用實驗 (L-carnitine/Choline assay) - 17 - 3.10氘標記膽鹼與肉鹼之代謝利用實驗 (d9-L-carnitine / d9-Choline assay) - 18 - 3.11競爭實驗 (competing assay) - 18 - 3.12電穿孔 (electroporation) - 18 - 第四章、 討論 - 19 - 第五章、未來展望 - 21 - 第六章、參考文獻 - 48 - 附錄一、 - 52 - | |
dc.language.iso | zh-TW | |
dc.title | 建立腸道菌叢培養體系統及分離一株低三甲胺產生者相關菌種 | zh_TW |
dc.title | Development of a culturomic system and isolation of a bacterial strain associated with low trimethylamine (TMA) producing phenotype | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 莊依萍(Yi-Ping Chuang),董馨蓮(Shin-Lian Doong) | |
dc.subject.keyword | 動脈粥樣硬化,冠狀動脈疾病,腸道微生物群,L-肉鹼,培養, | zh_TW |
dc.subject.keyword | Atherosclerosis,Coronary artery disease,Gut microbiota,L-carnitine,culturomics, | en |
dc.relation.page | 52 | |
dc.identifier.doi | 10.6342/NTU201901789 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2019-07-22 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 微生物學研究所 | zh_TW |
dc.date.embargo-lift | 2022-08-28 | - |
顯示於系所單位: | 微生物學科所 |
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