Wilson病患者肠道微生物多样性及其与临床相关性分析

发布时间：2018-02-09 02:55

本文关键词： Wilson病肠道微生态拟杆菌微量元素 UWDRS　出处：《安徽中医药大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的1.1基于Illumina PE250测序平台对Wilson病患者和健康人群肠道微生物16S rRNA基因V4高变区进行测序分析;利用生物信息学分析方法对已测基因序列进行数据统计与分析,观察Wilson病肠道微生物的多样性及结构特点。1.2比较Wilson病患者肠道细菌丰度与Wilson病患者临床指标方面的相关性,探索肠道微生态失衡在本病发生发展中的作用机制。方法2.1选取22例首诊Wilson病患者作病例组,选取22例健康人为正常对照组,并收集入组对象的粪便标本;2.2运用柱式粪便DNAout试剂盒抽提入组对象粪便菌群DNA,随即进行PCR扩增、Illumina PE250文库构建及16S rRNA基因V4高变区测序;2.3运用免疫比浊法检测Wilson病患者铜生化;运用火焰原子吸收光谱法(FAAS)测定Wilson病患者血清及粪便微量元素(铜、铁、锌)。结果3.1与正常对照组相比,WD病例组肠道微生物群落Shannon指数显著降低(2.61 vs 2.53,P0.05)3.2从门(Phylum)水平上分析可知,WD病例组肠道的Proteobacteria(4.31%vs 3.09%,P0.001),Tenericutes(0.23%vs 0.19%,P0.001)和Fusobacteria(1.88%vs 0.04%,P0.001)及Firmicutes(26.18%vs 19.83%,P0.001)细菌丰度较正常对照组显著增加;而Bacteroidetes丰度显著减少(67.19%vs 76.75%,P0.001);3.3在属(Genus)的水平上,WD病例组的肠道微生物中Bacteroides(48.5%vs 4.6%,P0.001),Faecalibacterium(2.9%vs 2.1%P0.001),Megamonas(0.8%vs 0.2%P0.001),Roseburia(0.7%vs 1.3%,P0.001)的丰度显著高于正常对照组;而Prevotella(1.6%vs 2.4%P0.001)的丰度明显减少;3.4 WD病例组的粪便Cu、Fe、Zn微量元素含量显著低于正常对照组。(Cu,T=-3.816,P0.001;Fe:T=-5.289,P0.001,Zn:T=-2.704,P0.001);3.5在门(phylum)水平上,Wilson病患者肠道内Firmicutes丰度与粪便Cu浓度呈负相关(R=-0.044,P=0.0410.05),Bacteroidetes丰度与粪便Cu浓度正相关(R=0.552,P=0.0080.01);3.6在属(genus)水平上,Wilson病患者肠道内Bacteroidetes丰度与评分负相关(R=-0.571,P=0.0060.01)结论4.1 Wilson病患者肠道微生物多样性较正常人群显著降低;4.2在门、属层面上Wilson病患者的肠道微生物结构与正常人群有显著不同;4.3 Wilson病患者肠道内Firmicutes、Bacteroidetes丰度与其粪Cu含量相关;4.4 Wilson病患者肠道内Bacteroidetes丰度可能与患者临床症状严重程度有关。
[Abstract]:Objective to sequence and analyze the V4 hypervariable region of 16s rRNA gene of intestinal microorganism in patients with Wilson disease and healthy population based on Illumina PE250 sequencing platform, and to analyze the sequence of detected genes by bioinformatics analysis. To observe the diversity and structural characteristics of intestinal microorganism in Wilson's disease. 1.2 to compare the relationship between intestinal bacterial abundance and clinical indexes in patients with Wilson's disease. To explore the mechanism of intestinal microecological imbalance in the occurrence and development of the disease, 22 patients with Wilson's disease were selected as the case group and 22 healthy persons as the normal control group. The fecal specimens were collected and extracted into the fecal microflora using column stool DNAout kit. The library of Illumina PE250 was amplified by PCR, and the 16s rRNA gene V4 hypervariable region sequencing 2.3 was used to detect the copper biochemistry of Wilson patients by immunoturbidimetry. Determination of Trace elements (Cu, Fe) in Serum and feces of patients with Wilson's Disease by Flame Atomic absorption Spectrometry. Results compared with the normal control group, the Shannon index of intestinal microbe community was significantly decreased by 2.61 vs 2.53P 0.05 / 3.2 compared with the normal control group. The results showed that the intestinal Proteobacteria(4.31%vs 3.09 P0.001Tenericutestio 0.23 and Fusobacteria(1.88%vs 0.04P0.001) and Firmicutes(26.18%vs 19.83 P0.001) were significantly increased in the WD case group as compared with the normal control group (P < 0.05). The results showed that the bacterial abundance of the intestinal tract was significantly higher in the WD group than that in the control group (P < 0.05). The results showed that the bacterial abundance of the intestinal tract was significantly higher in the WD case group than in the normal control group (P 0.001) and Fusobacteria(1.88%vs 0.04 P 0.001 (P 0.001). The abundance of Bacteroidetes in intestinal microorganism of WD group was significantly lower than that of normal control group (P < 0.01). The abundance of Bacteroides(48.5%vs in intestinal microbe of WD group was significantly higher than that of normal control group (P < 0.01). The abundance of Bacteroides(48.5%vs in the intestinal microbe of WD group was significantly higher than that in normal control group (P < 0.01). The abundance of Bacteroides(48.5%vs in the intestinal microbe of WD group was significantly higher than that in the control group. The abundance of Bacteroides(48.5%vs in the stomach was significantly higher than that in the normal control group (P < 0.01), and the abundance of Bacteroides(48.5%vs 4.6C P 0.001P 0.001T 2.9vs 2.1P 0.001P 0.001a 0.8 vs 0.2P 0.001N was significantly higher than that of the normal control group. The abundance of Prevotella(1.6%vs 2.4P0.001) in feces of patients with Wilson's disease was significantly lower than that in normal control group (P 0.001). The contents of trace elements in feces of patients with Wilson's disease were significantly lower than those in the control group. The concentrations of Firmicutes in intestinal tract were negatively correlated with the concentration of fecal Cu in patients with Wilson's disease at the level of 2.4p0.001. The levels of Firmicutes were negatively correlated with the concentrations of fecal Cu in patients with Wilson disease at the level of Prevotella(1.6%vs 2.4P0.001. The concentrations of Firmicutes in the intestine were negatively correlated with the concentrations of fecal Cu in patients with Wilson's disease at the level of 2.4p0.001. The contents of trace elements in feces were significantly lower than those in the normal control group. The concentrations of Firmicutes in the intestinal tract were negatively correlated with the concentrations of fecal Cu in patients with Wilson's disease. There was a negative correlation between the intestinal Bacteroidetes abundance and the score of Bacteroidetes in the patients with Wilson's disease. Conclusion the intestinal microbial diversity of 4.1 Wilson patients was significantly lower than that of the normal controls. The intestinal microorganism structure of patients with Wilson's disease was significantly different from that of normal controls. The intestinal abundance of Firmicutesus Bacteroidetes in patients with Wilson's disease was correlated with their fecal Cu content. Bacteroidetes abundance in the intestine of patients with Wilson's disease might be related to the severity of clinical symptoms.
【学位授予单位】：安徽中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R742.4

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