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標題: | 建立培養體系統及分離高三甲胺產生表型之相關菌種 Development of a culturomic system and isolation of bacterial species associated with high trimethylamine(TMA) producing phenotype |
作者: | Ching-Yu Chiu 邱淨妤 |
指導教授: | 王錦堂(Jin-Town Wang) |
關鍵字: | 冠狀動脈疾病,腸道菌叢,Ruminococcus bicirculans,左旋肉鹼,氧化三甲胺, Coronary artery disease,Gut microbiota,Ruminococcus bicirculans,L-carnitine,Trimethylamine-N-oxide, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 目前已知腸道菌叢(gut microbiota)會影響人類健康,最近也有報告顯示腸道菌叢與冠狀動脈疾病(coronary artery disease, CAD)相關,因此我們想建立腸道菌叢的培養體系統(culturomic system)以了解冠狀動脈疾病。
我們利用高登培養基(Gordon’s medium)以及伊藤培養基(Itoh’s medium)的5種非選擇性培養基(non-selective medium)和11種選擇性培養基(selective medium)進行健康人腸道菌叢之培養,發現高登培養基及非選擇性培養基可培養出較多菌種。再利用選擇性培養基分離出有興趣的菌種,結果分離菌種效率只有60%以下。另外,比較檢體收集後的貯存方式,結果發現冷凍檢體可培養出新鮮檢體30%-70%菌種。以細菌16S核醣體DNA定序(bacterial 16S ribosomal DNA sequencing)鑑定菌種,發現比基質輔助雷射脫附游離飛行時間質譜儀(MALDI-TOF)的鑑定效果佳。 我們的合作實驗室研究發現,給予健康受試者左旋肉鹼(L-carnitine)後,挑選出14位在血漿中測量到較高濃度的氧化三甲胺(trimethylamine-N-oxide, TMAO),之後利用細菌16S核醣體DNA定序分析,發現其腸道菌叢中有較高比例的瘤胃球菌屬(Ruminococcus)、毛螺菌科(Lachnospiraceae)、柔膜菌綱(Mollicutes)腸道菌。他們也利用宏基因組定序(shotgun metagenomic sequencing)方式,發現Odoribacter splanchnicus、Ruminococcus bicirculans、Bacteroides cellulosilyticus高度存在於TMAO濃度較高的健康受試者腸道菌叢中,且三者皆帶有cutC、cutD基因。 由於無法培養分離Ruminococcus bicirculans,為了釐清CutC和CutD蛋白質是否能夠將左旋肉鹼代謝成三甲胺(trimethylamine, TMA),將Ruminococcus bicirculans的cutC和cutD基因克隆到大腸桿菌並表現。萃取V14受試者糞便中的gDNA,以此作為模板進行聚合酶連鎖反應(PCR),將Ruminococcus bicirculans的cutC和cutD基因放大後接在表現載體pET28c上並轉型到大腸桿菌BL21(DE3),結果發現Ruminococcus bicirculans的CutC和CutD無法將d9-choline、d9-carnitine代謝產生γ-丁基甜菜鹼(γ-butyrobetaine, γBB)或TMA。 總結,冷凍方式貯存糞便檢體為方便且是檢體收集的替代方法;高登培養基及伊藤非選擇性培養基為較好的腸道菌叢培養基;細菌16S核醣體DNA定序是準確性高的方法。以大腸桿菌表現Ruminococcus bicirculans的cutC和cutD並無功能。 It is currently known that gut microbiota affects human health, and recent research have shown that intestinal flora is associated with coronary artery disease (CAD). Therefore, we aim to establish a culturomic system of human gut microbiota to understand the association of human gut microbiota with CAD. We cultured human gut microbiota from healthy volunteer’s stools by Gordon's medium and Itoh's medium which included 5 non-selective media and 11 selective media. The result showed that Gordon's medium and non-selective media cultivated more species than other did. Next, we used selective media to isolate specific species from cultivated gut microbiota, the efficiency of isolation is below 60%. In addition, comparing the storage methods for stool sample, we found that the culture rate from the frozen sample was 30%-70% compared with that from the fresh sample. Bacterial 16S ribosomal DNA sequencing is better than matrix-assisted laser desorption free time-of-flight mass spectrometer (MALDI-TOF) for the bacterial species identification. Our co-laboratory found that healthy subjects challenged with L-carnitine, 14 of them with high concentrations of trimethylamine-N-oxide (TMAO) in plasma were detected, and a high proportion of Ruminococcus, Lachnospiraceae, Mollicutes in their gut microbiota were analyzed by bacterial 16S ribosomal DNA sequencing. Moreover, they also found a high proportion of Odoribacter splanchnicus, Ruminococcus bicirculans, Bacteroides cellulosilyticus and all of them harbored cutC and cutD genes in high TMAO producers’ gut microbiota by shotgun metagenomic sequencing. We are unable to culture and isolate Ruminococcus bicirculans. To delineate whether the CutC and CutD proteins were able to metabolize L-carnitine to trimethylamine(TMA), the cutC and cutD genes of Ruminococcus bicirculans were cloned and expressed in Escherichia coli. The gDNA from the feces of the V14 subject was extracted and then as the template for PCR. The cutC and cutD genes of Ruminococcus bicirculans were amplified and ligated into the expression vector pET28c and transformed to E. coli BL21 (DE3). Our results found that CutC and CutD of Ruminococcus bicirculans were not able to metabolize d9-choline and d9-carnitine to γ-butyrobetaine (γBB) or TMA. In summary, frozen stool sample is a convenient and an alternative method for sample collections; Gordon’s medium and Itoh’s non-selective media are the better media for culture of human gut microbiota; bacterial 16S ribosomal DNA sequencing is an accurate method for identification. The cutC and cutD genes of Ruminococcus bicirculans have no function in E. coli BL21 (DE3). |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78752 |
DOI: | 10.6342/NTU201901050 |
全文授權: | 有償授權 |
顯示於系所單位: | 微生物學科所 |
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