| Part 1 The inhibiton effect of probiotics on ulcerative colitis carcinogenesis and analysis of intestinal microbiotaObjective:To investigate the effects of probiotic VSL#3 and Saccharomyces boulardii on tumor formation of the AOM/DSS-induced mice model,and analyze the changes of fecal and intestinal mucosal microbiota to explore the role of intestinal microbiota in UC carcinogenesis.Methods:C57BL/6 mice were randomly divided into 7 groups.Group 1-6 were designed as ulcerative colitis carcinogenesis model.On the first day of week one,they were given intraperitoneal injection of 12.5mg/kg AOM.1 week later,they were given 2.5%DSS drinking water for 5 days,then replaced with ordinary drinking water.Group 1-5 were lavaged respectively with 5-AS A(75mg/kg/d),Saccharomyces boulardii(5x107CFU/d),VSL#3(1.5x109CFU/d),5-ASA combined with Saccharomyces boulardii,and 5-AS A combined with VSL#3 from the day of injection AOM(5 days/week).Group 6 was model control group,no gavage intervention was given.Group 7 was blank control group,no modeling and gavage intervention was given.After the mice sacrificed at the end of week twelve,we compare the tumor load in each group.The stool and intestinal mucosa samples were collected to analyze the differences of intestinal microbiota by 16s rDNA sequencing method.Results:1.The tumor formation rate:63-85%in the intervention group and 100%in the model control group.2.The tumor load:the tumor load in 5-ASA group(0.43±0.14cm),Saccharomyces boulardii group(0.20 ± 0.07cm),VSL#3 group(0.25 ± 0.07cm),5-ASA+ Saccharomyces boulardii group(0.53 ± 0.11 cm),5-ASA + VSL#3 group(0.46 ±0.11 cm)were significantly lower than those in the control group(0.97±0.19cm)(P=0.0269,p = 0.0006,p = 0.0009,p = 0.0486,p = 0.0261).3.Analysis of stool microbiota:(1)The model control group was lower in the Lactobacillus,and higher in Oscillibacter and Lachnoclostridium than in the blank control group.(2)After the intervention of probiotics,Bacillus,Lactococcus,Lactobacillus were increased,and Lachnoclostridiu,Oscillibacter,Bacteroides,Pseudomonas were reduced.4.Analysis of intestinal mucosal microbiota:(1)The model group was lower in Bifidobacterium and Ruminococcaceae_UCG-014,and higher in Alloprevotella.(2)Compared with the model control group,Lachnoclostridium and Lachnospiraceae_NK4A136 were increased in Saccharomyces boulardii group;Bifidobacterium were increased in VSL#3 group.Conclusion:1.VSL#3 and Saccharomyces boulardii can significantly reduce the tumor formation rate and tumor burden of UC carcinogenesis mice.2.The intestinal microbiota dysbiosis exists in UC carcinogenesis mice,beneficial genus reduced and harmful genus increased in both mucosal and fecal microbiota.3.Supplementary probiotics is beneficial to fecal and mucosal intestinal of UC carcinogenesis mice to achieve rebalancing.4.Bifidobacterium is easy to colonize in intestinal mucosa,Bacillus and Lactococcus are easily to colonized in feces.Part 2 Effect and mechanism of inflammatory factors and Saccharomyces boulardii on Wnt signaling pathwayObjective:To investigate the effect and mechanism of IL-6,TNF-a and Saccharomyces boulardii on Wnt signaling pathway.Methods:CCC-HIE-2,Caco-2 and HCT-116 were stimulated with IL-6 and TNF-αrespectively.The activity of Wnt signaling pathway was detected by double luciferase reporter assay.The effect of PDTC which was the inhibitor of NF-xB on the regulation of IL-6 and TNF-a in Wnt signaling pathway was observed.Co-culture Saccharomyces boulardii with cells to investigate the effects on the regulation of IL-6 and TNF-a in Wnt signaling pathway.Results:1.The stimulation by different concentration of IL-6 and TNF-a on CCC-HIE-2,Caco-2 and HCT-116 can increase the relative luciferase activity and up-regulate Wnt signaling pathway.And the best stimulation concentration and time were 50ng/ml and 6h.2.PDTC can inhibit the effect of up-regulation of relative luciferase activity by IL-6 and TNF-a on CCC-HIE-2(1.044±0.006 vs 0.895±0.003 p=0.0032,1.126±0.006 vs 1.062±0.014 p=0.0006),Caco-2(8.859±0.050 vs 7.922±0.075 p=0.0046,3.951±0.006 vs 3.550±0.02 p=0.0016)and HCT-116(2.728+0.053 vs 2.430±0.022 p=0.0008,4.044±0.022 vs 3.629±0.025 p=0.0033).3.Saccharomyces boulardii can inhibit the effect of up-regulation of relative luciferase activity by IL-6 and TNF-a on CCC-HIE-2(1.239±0.015 vs 0.956±0.001 p=0.0014,1.022±0.014 vs 0.780±0.026 p=0.0074),Caco-2(9.731±0.075 vs 8.479±0.099 p=0.0049,9.187±0.044 vs 7.691±0.055 p=0.0011)and HCT-116(4.116±0.004 vs 3.734±0.045 p=0.0068,4.489±0.010 vs 3.858±0.021 p=0.0007).Conclusion:1.TNF-a and 1L-6 can up-regulate the Wnt signaling pathway through NF-(?)B pathway on CCC-HIE-2、Caco-2、HCT-116.2.The up-regulation of Wnt signaling pathway by TNF-a and IL-6 can be inhibited by Saccharomyces boulardii co-cultured with CCC-HIE-2、Caco-2、HCT-116.Part 3 Analysis of intestinal microbiota in patients with ulcerative colitis of different lesion rangeObjective:To investigate the characteristics of intestinal microbiota in patients with ulcerative colitis of different lesion range.Methods:Stool sample of 10 healthy individuals,stool and intestinal mucosa sample of 35 UC patients(7 cases of rectal type,9 cases of left colon type and 19 cases of extensive colon type)were collected to analyze the intestinal microbiota differences by 16s rDNA sequencing method.Results:1.Analysis of fecal microbiota:(1)The diversity of intestinal microbiota was significantly lower in extensive colon type UC patients than in healthy individuals.(2)Compared with healthy individuals,Subdoligranulum,Ruminococcus-2,Fusicatenibacter,Faecalibacterium,Pseudobutyrivibrio and Alistipes were reduced in UC patients.Escherichia-Shigella,Peptoclostridium and Veillonella were increased.(3)Rectal type UC patients possessed more Pseudobutyrivibrio than left colon type UC patients,and more Lachnospira,less Prevotella-9 than extensive colon type UC patients.2.Analysis of mucosal microbiota:(1)Compared with rectal type patients,Lactococcus,Geobacillus,Streptococcus and Leuconostoc were decreased in left colon and extensive colon type patients.(2)Compared with extensive colon type patients,rectal and left colon type patients possessed less Bacteroides.(3)There was no significant difference in the biological diversity and bacterial composition between the left and right mucosal microbiota in all types of UC patients.(4)The biological diversity of mucosal microtiota in UC patients was higher than that of fecal microbiota,and their composition was different.Conclusion:1.The biological diversity of fecal microbiota in UC patients is reduced,as the number of beneficial genus is reduced and the harmful genus is increased.2.With the expansion of the lesion,both fecal and mucosal microbiota posess less beneficial and more harmful genus.3.Changes in mucosal microbiota of UC patients distribute in the whole colon,not limited to the lesion mucosa.4.There are significant differences of biological diversity and composition between fecal and mucosal microbiota in UC patients.Objective:To enhance the clinicians’ understanding of gastric neuroendocrine neoplasms by assessing the clinical features of these patients.Methods:A total of 36 patients with GNENs who were diagnosed between October 2005 and October 2015 at Peking Union Medical College Hospital were retrospectively reviewed.The demographic characteristics,clinical manifestations,endoscopic appearance and pathologic features as well as the treatments of GNENs were collected and analyzed.Results:1.The average age of 36 patients was 55.8 years,male to female ratio was 0.89:1.2.The clinical manifestations varied,in which abdominal pain was as high as 42.9%.3.63.9%of the lesions were distributed in the gastric body.The endoscopic appearance of GNENs included polypoid lesions,ulcerative lesions,mucosal depression.Polypoid lesions was the most common,and 73.9%of these polyps were single,with an average diameter of less than 10mm.4.Pathological grading included G1-G3 level,while G1 level accounted for 55.6%.5.65.2%patients who undergone gastric body biopsy had pathologic evidence of mucosa atrophy or metaplasia.6.Therapeutic modalities included endoscopic intervention and surgical resection.Conclusion:1.Patients with GNENs normally presented with non-specific symptoms.The most common endoscopic appearance was polypoid lesions,in gastric body.2.Clinicians should be aware of the possibility of GNENs in the polypoid lesions,particularly for those accompanied with gastric body atrophy. |
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