Research On Intestinal Microbial Barrier Of Ulcerative Colitis And Ileal Pouch

Objective: To analyze intestinal dominant bacterial species of the patients with ulcerative colitis(UC) and ileal pouch anal anastomosis(IPAA), and to explore the mechanism of microbiology mechanism of UC and pouchitis.Methods: Fecal samples were collected from patients with UC and IPAA who were treated in Tianjin Medical University General Hospital from July 2013 to July 2014. According to “Mayo Scoring System for Assessment of Ulcerative Colitis Activity”, the patients with UC were divided into four groups: remission, mild activity, moderate activity and severe active. According to “Pouchitis Disease Activity Index(PDAI)”, the patients with IPAA were divided into two groups: <7 points and ≥7 points. To extract DNA from fecal samples in using the genomic DNA extraction kit. Designed for V3 region’s universal primers with GC fragment of bacterial 16 Sr DNA gene. Touchdown PCR technique was used to amplify fecal genomic DNA. The PCR products were analyzed with diversity and similarity cluster analysis by denaturing gradient gel electrophoresis(DGGE). Finally, the DGGE gels were cut plastic, recycling and sequenced to verify the bacterial species.Results: Control group: Intestinal bacterial composition is complex, different from each other and the complexity of the intestinal bacteria also represents the complexity of intestinal microbial barrier. DGGE profiles of 16 Sr DNA V3 region gene of healthy human mainly include 16.63±2.872 main advantage bands. The main advantage bacteria of healthy human are Bacteroides spp.. UC groups: Compared with control group, the diversity of intestinal bacteria of UC patients is decreased and the trend is more obvious with the disease become serious, which has statistically significant difference(p <0.05). Compared with control group, intestinal bacterial species of UC patients has changed, mainly include beneficial bacteria such as Escherichia are induced, and harmful bacteria such as pathogenic Escherichia coli are increased. Dice similarity analysis and UPGMA tree analysis show that differences within groups are less than differences between groups which has statistically significantdifference(p<0.05). PCA analysis shows that the amount of the first two ordination axes explain species variable is 40.3%, different groups can be separated from each other. IPAA groups: Compared with control group, the ileal pouch normal flora of IPAA patients changed, ileal pouch bacterial diversity of IPAA patients decrease and the trend is more obvious with the score of PDAI increased, which has statistically significant difference(p<0.05). Dice similarity analysis and UPGMA tree analysis show that differences within groups are less than differences between groups which has statistically significant difference(p<0.05). PCA analysis shows that the amount of the first two ordination axes explain species variable is 40.0%, different groups can be separated from each other.Conclusion: The quantity and species of intestinal bacteria of patients with ulcerative colitis have changed significantly. Results of this experiment provide an basis on the therapy of probiotics, antibiotics and fecal transplantation, and also provide the basis for the next step by specific bacteria enema and construct animal model. Ileal pouch normal flora has changed significantly, patients with pouch pouchitis have a flora disorder. Due to the interactions between intestinal flora and pouch mucosal or intestinal mucosal, intestinal and pouch mucosal damage.