本文關鍵詞： Wilson病 腸道微生態(tài) 擬桿菌 微量元素 UWDRS　出處：《安徽中醫(yī)藥大學》2016年碩士論文　論文類型：學位論文

【摘要】：目的1.1基于Illumina PE250測序平臺對Wilson病患者和健康人群腸道微生物16S rRNA基因V4高變區(qū)進行測序分析;利用生物信息學分析方法對已測基因序列進行數(shù)據(jù)統(tǒng)計與分析,觀察Wilson病腸道微生物的多樣性及結(jié)構(gòu)特點。1.2比較Wilson病患者腸道細菌豐度與Wilson病患者臨床指標方面的相關性,探索腸道微生態(tài)失衡在本病發(fā)生發(fā)展中的作用機制。方法2.1選取22例首診Wilson病患者作病例組,選取22例健康人為正常對照組,并收集入組對象的糞便標本;2.2運用柱式糞便DNAout試劑盒抽提入組對象糞便菌群DNA,隨即進行PCR擴增、Illumina PE250文庫構(gòu)建及16S rRNA基因V4高變區(qū)測序;2.3運用免疫比濁法檢測Wilson病患者銅生化;運用火焰原子吸收光譜法(FAAS)測定Wilson病患者血清及糞便微量元素(銅、鐵、鋅)。結(jié)果3.1與正常對照組相比,WD病例組腸道微生物群落Shannon指數(shù)顯著降低(2.61 vs 2.53,P0.05)3.2從門(Phylum)水平上分析可知,WD病例組腸道的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)細菌豐度較正常對照組顯著增加;而Bacteroidetes豐度顯著減少(67.19%vs 76.75%,P0.001);3.3在屬(Genus)的水平上,WD病例組的腸道微生物中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)的豐度顯著高于正常對照組;而Prevotella(1.6%vs 2.4%P0.001)的豐度明顯減少;3.4 WD病例組的糞便Cu、Fe、Zn微量元素含量顯著低于正常對照組。(Cu,T=-3.816,P0.001;Fe:T=-5.289,P0.001,Zn:T=-2.704,P0.001);3.5在門(phylum)水平上,Wilson病患者腸道內(nèi)Firmicutes豐度與糞便Cu濃度呈負相關(R=-0.044,P=0.0410.05),Bacteroidetes豐度與糞便Cu濃度正相關(R=0.552,P=0.0080.01);3.6在屬(genus)水平上,Wilson病患者腸道內(nèi)Bacteroidetes豐度與評分負相關(R=-0.571,P=0.0060.01)結(jié)論4.1 Wilson病患者腸道微生物多樣性較正常人群顯著降低;4.2在門、屬層面上Wilson病患者的腸道微生物結(jié)構(gòu)與正常人群有顯著不同;4.3 Wilson病患者腸道內(nèi)Firmicutes、Bacteroidetes豐度與其糞Cu含量相關;4.4 Wilson病患者腸道內(nèi)Bacteroidetes豐度可能與患者臨床癥狀嚴重程度有關。
[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.
【學位授予單位】：安徽中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R742.4

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