人體腸道菌相與其在臨床上的一個應用

Human Intestinal Microflora and Its Clinical Application

廖家敏(Chia-Min Liao)

指導教授 ： 于宏燦

國立臺灣大學/生命科學院/動物學研究研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.00778

摘要

人體的腸道中分佈著數量龐大且複雜的微生物群落，然而，大部份的腸道微生物以傳統培養的方式與技術仍難以得之。目前大部份微生物學家均以細菌的小單元核糖體RNA基因(16S rDNA)為主，藉由不同的分析方法，如：ARDRA (RFLP)、DGGE、TGGE、T-RFLP、FISH，等；用以區分不同菌種之遺傳變異與組成，以作為細菌分類的依據。

本研究利用分子生物學的方法來探究人體的腸道細菌，分析其組成與變異，用以建立一個人體腸道細菌相藍圖，將來可應用於臨床醫學上與人體相關之腸胃道疾病與細菌感染之研究上。因此，本研究取得一個正常人的糞便檢體，並設計普遍性引子(universal primer)，行聚合酶鏈鎖反應(PCR)以放大細菌之16S rDNA片段，再利用轉殖(cloning)的方式，複製轉殖之16S rDNA片段。再隨機選擇序列進一步作定序分析，藉由比對序列之相似性，用以建立親緣關係樹，以作為細菌組成之分析與分類之依據，並建構一個人體腸道菌相之16S rDNA基因資料庫。所得序列間有98%的相似度者，則為歸為同一操作型分類單元(OTU)或單型(phylotype)。本研究所得之259條序列或clones共可分成11個群，共占細菌界(domain Bacteria)之四個門(phylum)，主要為：Firmicutes (49.8%)、Bacteroidetes (40.9%)、Proteobacteria (8.9%)與Verrucomicrobia (0.4%)。其中有15個clones (16.7 %)為新的單型，多數為已知之菌種。

臨床研究上，許多人體之感染性疾病，均與腸道細菌的轉移有相關。在此，為探究肝硬化病人併發自發性腹膜炎之致病機制，取得病人之血液與腹水檢體，並與正常人之血液檢體做對照，並利用聚合酶鏈鎖反應與無性繁殖的方式，檢測其血液與腹水中潛在之細菌。最終取得100條序列，進一步作親緣關係之分析結果，所得之菌種可分成三大群，主要以α-proteobacteria, β- proteobacteria與γ-proteobacteria為主。並包含Deinococci一小支分群。此外，值得關切的是正常人的血液中含有Stenotrophomonas maltophilia之菌種。而肝硬化併發腹水之病患的血液與腹水中各含有不同的菌種，如：Ralstonia gilardii、Burkholderia symbiont、Blastobacter denitrificans、Acinetobacter johnsonii、Ralstonia insidiosa等相關菌種。而在臨床研究上亦發現S. maltophilia與B. denitrificans等相關菌種具有抗藥性，易造成重症病人感染之症狀。

關鍵字

人體腸道菌 ； 小單元核糖體DNA ； 自發性細菌感染腹膜炎

並列摘要

There are numbers of microflora incubating at human intestinal tract, but there are still 60-80% microbes can't be cultivated in vitro.Using 16S rDNA as a genetic marker and different analytic method, such as:ARDRA (RFLP), DGGE, TGGE, T-RFLP, FISH,etc. to investigate the composition of microflora and classification will be the modern method.
This study will use molecular method to investigate the human intestinal microflora composition and its clinical application. Using universal primer and PCR to amplify bacterial 16S rDNA in human fecal sample, random cloning and sequencing to obtain the sequences.Sequence similarity higher than 98% will be the same OTU or phylotype. Final obtained 259 sequences can be classified into 11 groups, 4 phyla:Firmicutes (49.8%)、Bacteroidetes (40.9%)、Proteobacteria (8.9%) and Verrucomicrobia (0.4%).There are 15 sequences (16.7%) will be novel phylotype, most sequeces are known.
There are many clinical infectious diseases related to human intestinal bacterial translocation. In order to investigate the pathogenesis of spontaneous bacterial peritonitis, we obtained patients blood and ascites to co-operate with PCR and random cloning to screen the bacterial DNA. Finally obtained 100 bacterial sequences and can be classified into 3 groups,α-proteobacteria, β- proteobacteria and γ-proteobacteria respectively.In addition, we also found that there are bacterias in health person's blood, mainly Stenotrophomonas maltophilia and other species, such as Ralstonia gilardii、Burkholderia symbiont、Blastobacter denitrificans、Acinetobacter johnsonii、Ralstonia insidiosa.

並列關鍵字

human intestinal microflora ； 16SrDNA ； SBP

參考文獻

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