本文選題：熒光定量 + PCR　； 參考：《江南大學(xué)》2012年碩士論文

【摘要】：硫酸鹽還原菌(sulphate-reducing bacteria, SRB)是一類能還原硫酸鹽產(chǎn)生H2S的厭氧菌，其中脫硫弧菌屬(Desulfovibrio, DSV)是人體結(jié)腸SRB的優(yōu)勢菌群。由于內(nèi)源性的H2S會毒害腸道上皮細(xì)胞，不少研究者推測DSV與腸道疾病之間存在一定的聯(lián)系，但目前國內(nèi)外的研究中，DSV與腸道疾病的關(guān)系還存在爭議。本研究從江蘇省無錫市第四人民醫(yī)院采集息肉、潰瘍性結(jié)腸炎、直腸結(jié)腸癌患者和健康人群的腸道內(nèi)容物，分析不同人群腸道中DSV的數(shù)量差異及其與年齡和性別的關(guān)系，結(jié)合不同人群腸道菌群和DSV的多樣性，探索DSV及腸道菌群多樣性與腸道疾病之間的潛在關(guān)系，對預(yù)防腸道疾病、揭示其致病機理和研究治療腸道疾病的相關(guān)藥物具有一定的意義。 本研究共采集58名受試者的腸道內(nèi)容物，其中直腸結(jié)腸癌患者15人，息肉患者15人，潰瘍性結(jié)腸炎患者13人和腸道健康人群15人。使用Blood DNA kit提取腸道細(xì)菌總DNA，并以其為模板，擴增DSV特異性基因片段，采用實時熒光定量PCR(RT-PCR)的方法，對受試者腸道DSV的數(shù)量進行定量，并分析DSV的數(shù)量與年齡、性別及腸道疾病之間的潛在關(guān)系。以腸道細(xì)菌總DNA為模板，采用巢式-PCR擴增GC-dsrB和GC-16S rRNAV3基因片段，采用變性梯度凝膠電泳(PCR-DGGE)技術(shù)，對不同人群的腸道DSV和腸道菌群結(jié)構(gòu)進行分析，選取優(yōu)勢條帶，結(jié)合16S rRNA V3區(qū)測序分析不同人群腸道菌群多樣性的差異。 RT-PCR分析顯示，，所有受試者均為DSV陽性，其中息肉和潰瘍性結(jié)腸炎患者腸道中DSV的數(shù)量分別為2.9×106(±8.6×105) cfu/mL和1.2×106(±1.6×105) cfu/mL，明顯高于健康人群的7.0×105(±2.1×105) cfu/mL (p0.05)，而直腸結(jié)腸癌患者腸道中DSV的數(shù)量為6.8×105(±1.0×105)cfu/mL，與健康人群無明顯差異(p0.05)。且在4個人群中DSV的數(shù)量與人的年齡變化沒有相關(guān)性(p0.05)，在所研究的息肉患者中，男性患者腸道中DSV的數(shù)量高于女性患者(p0.05)，而在其他3個人群中無明顯差異(p0.05)。DGGE圖譜聚類分析結(jié)果表明，腸道疾病人群腸道中DSV的菌群相似度較高，而與健康人群之間的差異較大。16S rRNAV3區(qū)基因測序顯示腸道疾病人群與健康人群在腸道菌群多樣性和優(yōu)勢菌群方面均有明顯差異。 通過RT-PCR與DGGE相結(jié)合的方法，說明腸道DSV數(shù)量的增多是息肉和潰瘍性結(jié)腸炎疾病的一個重要特征，且其菌群組成在腸道疾病人群與健康人群之間存在明顯差異。與健康人群相比，腸道疾病人群的腸道微生物多樣性升高，優(yōu)勢菌群發(fā)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 (鹵8.6 脳 10 5) cfu/mL and 1.2 脳 10 6 (鹵1.6 脳 10 5) cfu-mL, respectively, which was significantly higher than that of healthy people (7.0 脳 10 5 (鹵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 (鹵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.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R378

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