不同人群脱硫弧菌数量及肠道菌群多样性分析

发布时间：2018-06-07 11:42

本文选题：荧光定量 + PCR　；参考：《江南大学》2012年硕士论文

【摘要】：硫酸盐还原菌(sulphate-reducing bacteria, SRB)是一类能还原硫酸盐产生H2S的厌氧菌，其中脱硫弧菌属(Desulfovibrio, DSV)是人体结肠SRB的优势菌群。由于内源性的H2S会毒害肠道上皮细胞，不少研究者推测DSV与肠道疾病之间存在一定的联系，但目前国内外的研究中，DSV与肠道疾病的关系还存在争议。本研究从江苏省无锡市第四人民医院采集息肉、溃疡性结肠炎、直肠结肠癌患者和健康人群的肠道内容物，分析不同人群肠道中DSV的数量差异及其与年龄和性别的关系，结合不同人群肠道菌群和DSV的多样性，探索DSV及肠道菌群多样性与肠道疾病之间的潜在关系，对预防肠道疾病、揭示其致病机理和研究治疗肠道疾病的相关药物具有一定的意义。本研究共采集58名受试者的肠道内容物，其中直肠结肠癌患者15人，息肉患者15人，溃疡性结肠炎患者13人和肠道健康人群15人。使用Blood DNA kit提取肠道细菌总DNA，并以其为模板，扩增DSV特异性基因片段，采用实时荧光定量PCR(RT-PCR)的方法，对受试者肠道DSV的数量进行定量，并分析DSV的数量与年龄、性别及肠道疾病之间的潜在关系。以肠道细菌总DNA为模板，采用巢式-PCR扩增GC-dsrB和GC-16S rRNAV3基因片段，采用变性梯度凝胶电泳(PCR-DGGE)技术，对不同人群的肠道DSV和肠道菌群结构进行分析，选取优势条带，结合16S rRNA V3区测序分析不同人群肠道菌群多样性的差异。 RT-PCR分析显示，，所有受试者均为DSV阳性，其中息肉和溃疡性结肠炎患者肠道中DSV的数量分别为2.9×106(±8.6×105) cfu/mL和1.2×106(±1.6×105) cfu/mL，明显高于健康人群的7.0×105(±2.1×105) cfu/mL (p0.05)，而直肠结肠癌患者肠道中DSV的数量为6.8×105(±1.0×105)cfu/mL，与健康人群无明显差异(p0.05)。且在4个人群中DSV的数量与人的年龄变化没有相关性(p0.05)，在所研究的息肉患者中，男性患者肠道中DSV的数量高于女性患者(p0.05)，而在其他3个人群中无明显差异(p0.05)。DGGE图谱聚类分析结果表明，肠道疾病人群肠道中DSV的菌群相似度较高，而与健康人群之间的差异较大。16S rRNAV3区基因测序显示肠道疾病人群与健康人群在肠道菌群多样性和优势菌群方面均有明显差异。通过RT-PCR与DGGE相结合的方法，说明肠道DSV数量的增多是息肉和溃疡性结肠炎疾病的一个重要特征，且其菌群组成在肠道疾病人群与健康人群之间存在明显差异。与健康人群相比，肠道疾病人群的肠道微生物多样性升高，优势菌群发生偏移，菌群失衡。
[Abstract]:Sulphate-reducing bacterium (SRB) is a kind of anaerobic bacteria which can reduce sulfate to produce H2S. Among them, Vibrio desulphurium belongs to Desulfovibrio (DSV), which is the dominant group of SRB in human colon. Because endogenous H2S can poison intestinal epithelial cells, many researchers speculate that there is a certain relationship between DSV and intestinal diseases, but the relationship between DSV and intestinal diseases is still controversial at home and abroad. In this study, the intestinal contents of polyps, ulcerative colitis, rectal colon cancer patients and healthy people were collected from the fourth people's Hospital of Wuxi City, Jiangsu Province. Combined with the diversity of intestinal flora and DSV in different populations, the potential relationship between DSV, intestinal flora diversity and intestinal diseases was explored to prevent intestinal diseases. It is of great significance to reveal the pathogenesis of the disease and to study the related drugs for the treatment of intestinal diseases. In this study, the intestinal contents of 58 subjects were collected, including 15 patients with rectal colon cancer, 15 patients with polyps, 13 patients with ulcerative colitis and 15 healthy people. The total DNA of intestinal bacteria was extracted by Blood DNA kit, and the specific gene fragment of DSV was amplified by using it as template. The quantity of DSV in intestinal tract was quantified by real-time fluorescence quantitative PCR RT PCR, and the number and age of DSV were analyzed. The underlying relationship between gender and intestinal diseases. Using total DNA of intestinal bacteria as template, GC-dsrB and GC-16S rRNAV3 gene fragments were amplified by nested polymerase chain reaction (NPCR), and denaturing gradient gel electrophoresis (DGGE) technique was used to analyze the structure of intestinal DSV and intestinal flora in different populations, and the dominant bands were selected. The diversity of intestinal flora in different populations was analyzed by sequencing 16s rRNA V3 region. RT-PCR analysis showed that all subjects were DSV positive. The number of DSV in the intestine of patients with polyps and ulcerative colitis was 2.9 脳 10 6 (卤8.6 脳 10 5) cfu/mL and 1.2 脳 10 6 (卤1.6 脳 10 5) cfu-mL, respectively, which was significantly higher than that of healthy people (7.0 脳 10 5 (卤2.1 脳 10 5) cfu/mL / ml), while the number of DSV in the intestine of patients with rectal colon cancer was 6.8 脳 10 5 (卤1.0 脳 10 5) cfumL, which was not significantly different from that of healthy people (p 0.05). Moreover, there was no correlation between the number of DSV and the age change of the population in 4 populations, and there was no correlation between the number of DSV and the age of the patients with polyp in the study. The number of DSV in intestinal tract of male patients was higher than that of female patients, but there was no significant difference among the other three populations. The results of cluster analysis showed that DSV in intestinal tract of patients with intestinal diseases had a higher similarity. The results of 16s rRNAV3 gene sequencing showed that there were significant differences in the diversity and dominance of the intestinal flora between the patients with intestinal diseases and the healthy people. The combination of RT-PCR and DGGE showed that the increase of intestinal DSV was an important characteristic of polyps and ulcerative colitis. Compared with the healthy population, the intestinal microorganism diversity of the intestinal disease population increased, the dominant flora shifted, and the bacterial flora was out of balance.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

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