| Constipation is the most common gastrointestinal disorder.Patients with refractory constipation can be divided into those who have a normal diameter gut and those who have gut dilatation.The latter include patients with Hirschsprung's disease,chronic idiopathic intestinal pseudoobstruction and idiopathic mega-colon.The etiology of refractory functional constipation and idiopathic mega-colon are unknown.Gut microbiota accommodates nearly 400 different species of bacteria,which plays a pivotal role in our health with activity including fermentation of dietary components,production of short-chain fatty acids,modulation of the immune system and epithelial gene expression,transformation of bile acids,production of vitamins and other health-protective substances,and provision of a barrier against pathogenic bacteria.Recently,more and more culture-independent methods,such as denaturing gradient gel electrophoresis(DGGE),Fluorescence in situ hybridization(FISH),DNA microarray and so on,were used to analyze the gut microbiota.Recent studies proved that altered microbial composition is involved in the initiation and maintenance of a number of gastrointestinal disorders,including antibiotic-associated diarrhea,irritable bowel syndrome,and inflammatory bowel disease.Gut microbiota is also believed to play a role in the initiation or maintenance of functional constipation,but studies on this is lacking.In patients with refractory constipation and idiopathic mega-colon,the content of colon stayed longer,so the harmful waste in colon also stayed longer.At the same time,the microbiota in colon was altered.All these changes gave more pressure for colonic barrier function.Tight junction and mucus layer are very important parts of barrier function.Therefore,we undertook the study(1)to characterize the changes of colonic mucosa-associated microbiota in relation to refractory constipation and idiopathic mega-colon using denaturing gradient gel electrophoresis(DGGE)method and(2)to observing the alteration of epithelial tight junction and mucus secretory cells--goblet cells.Part ?Alteration of Colonic Mucosa-associated Microbiota in Patients with Refractory Constipation and Idiopathic Mega-colonStudy ?.Dysbiosis of Colonic Mucosa-associated Microbiota in Patients with Refractory ConstipationAims:Our aim of this study was to investigate the characterization of colonic mucosa-associated microbiota in RC,and to identify individual bacterial species that contribute to the characterization.Materials and Methods:Sixteen RC patients and 9 controls were included in this study.Mucosal tissues from sigmoid colon were obtained during surgery.Mucosa-associated microbiota was analyzed by Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis(PCR-DGGE)and the predominant bacterial composition was determined with DNA sequencing.Results:Colonic mucosa-associated microbiota composition of RC patients was different from the controls.Band profiles revealed that numbers of predominant bacteria were significantly different between RC and control group.By assessing the mean band numbers in RC(18.38±5.20),compared to the controls(28.22±5.24),a significant decrease in bacterial species in RC was suggested.Biodiversity of the bacterial species was significantly lower in RC than the controls(P=0.01).The mucosa-associated bacterial dysbiosis of RC was characterized by a reduced proportion of Bacteroidetes and Firmicutes and an elevated abundance of two uncultured bacteria(P<0.05).Conclusion:Molecular profiling revealed abnormalities of colonic mucosa-associated microbiota in RC,which was characterized by a reduced proportion of Bacteroidetes and Firmicutes and an elevated abundance of two uncultured bacteria.Study ? Dysbiosis of Colonic Mucosa-associated Microbiota in Patients with Idiopathic Mega-colonAims:Our aim of this study was to investigate any quantitative differences in colonic mucosa-associated bacterial compositions in idiopathic mega-colon(MC),and to identify individual bacterial species that contribute to these differences.Methods:Ten MC patients and 11 controls were included in this study.Mucosal tissues from sigmoid colon were obtained.Mucosal microbiota was analyzed by Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis(PCR-DGGE)and the predominant bacterial composition in the mucosa was determined with DNA sequencing.Results:Band profiles revealed that number of predominant mucosa-associated bacteria were significantly different between MC group and control group(P<0.01).An increase in bacterial species in MC group was indicated.Shannon-Wiener diversity score was significantly higher in MC group compared with control group(P<0.01),suggesting that the biodiversity of mucosa-associated microbiota in patients with MC was increased.DNA sequencing suggested that some special bacteria in MC group distinguished from the controls.Conclusion:Molecular profiling revealed abnormalities of colonic mucosa-associated microbiota in MC.Biodiversity of colonic mucosal microbiota in patients MC was increased.The dysbiosis is characterized by an increase of the richness of 5 species of Bacteroidetes and a decrease in 1 species of Bacteroidetes.Part ?Alterations of Colonic Tight Junction and Goblet Cells in Patients with Refractory Constipation and Idiopathic Mega-colonStudy ?.Alterations of Colonic Tight Junction in Patients with Refractory Constipation and Idiopathic Mega-colonAim:The aim of the study is to explore the alterations of the ultrastructure of colonic tight junction and the expression of proteins of colonic tight junctions(Occludin and Claudin-1)in patients with refractory constipation(RC)and idiopathic mega-colon(MC).Patients and methods:Eight MC patients,8 RC patients and 8 controls were included in this study.Tissues from patients' sigmoid colon were obtained during the surgery.The pathological and ultrastructural changes were comparatively observed by light microscopy and scan electron microscopy.At the same time,the expression of Occludin and Claudin-1 were semiquantitatively evaluated with Western blotting and immunofluorescence techniques.Result:No obvious pathological was observed in RC and MC patients.The colonic tight junctions of RC and MC patients kept integrity.No significant difference was found in the expression of Occludin and Claudin-1 among patients in RC gourp,MC group and control group.Conclusion:Although the pressure of the colon in RC and MC patients were increased,the colonic gut barrier kept normal.Study ? Alterations of Goblet Cells in Patients with Refractory Constipation and Idiopathic Mega-colonAim:The aim of the study is to explore the alterations of goblet cells in patients with refractory constipation(RC)and idiopathic mega-colon(MC).Patients and methods:Eight MC patients,8 RC patients and 8 controls were included in this study.Tissues from patients' sigmoid colon were obtained during the surgery.The alteration of colonic epithelial microvili and goblet cells was comparatively observed by light microscopy.At the same time,goblet cells were observed by AB/PAS stain.Result:Compared with the control group,the colonic epithelial microvili of RC group and MC group were much thinner and MC group was thinner than RC group.The number of goblet cells per view under light microscopy(x40)was significantly larger in MC group and RC group,compared to the control.The number in MC group was larger than RC group.Conclusion:In patients with RC and MC,the colonic epithelial microvilli became thinner while the number of goblet cells became larger. |
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