Effect Of Pathogenic Bacteria On The Intestinal Barrier And Structural Proteins Of Guinea Pig

In modern life,people's eating habits is Irregular,an increasing number of food additives and antibiotics misuse,resulting in our gut barrier function is impaired,leading to bacterial breakthrough intestinal barrier into blood and invasion of the body organs.Our research group previously published articles,using clinical samples found bacteria exist in the bile of patients with acute cholecystitis,and isolation and identification of a strain of pathogenic Escherichia coli Enteroinvasive Escherichia coli and EIEC.So what is the cause of the disease is the way into the gallbladder to cause inflammation of it? This topic simulation antibiotics state of gut barrier is impaired,and by PCR-DGGE technique to identify the existence of the state,and packet processing animal model,a group with LGG gavage,to verify the antibiotic caused the intestinal barrier injury can be probiotic repair,intestinal structure if there is a change;another group of pathogenic Escherichia coli by gavage and the intestinal bacteria group diversity,and intestinal morphology change.Studies have shown that intestinal pathogenic bacteria can be by changing the tight junction barrier structure and function of protein,so that it is conducive to bacterial invasion and infection,so as to control the function of host cell body [5].These effects may lead to direct changes in tight junction proteins,such as Occludin2 and Claudin protein in this paper;By immunohistochemistry to analyze the intestinal tight junction protein expression,and detection of liver,blood whether it contains the pathogenic bacteria,and to explore the pathogenic bacteria by intestinal mucus consumption as carbon source,to break through the intestinal barrier into blood? Or the pathogen will gut barrier damage,which caused a large number of other bacteria into the blood into the liver,and finally into the gallbladder caused by cholecystitis? Objective:This thesis simulates the intestinal barrier damage caused by antibiotics,and the PCR-DGGE technology to identify the state of existence,then the packet processing model animal,a group with Lactobacillus rhamnosus gavage,to verify whether gut barrier injury caused by antibiotics can be repaired intestinal probiotics,whether the structure has changed;another group with pathogenic Escherichia coli by gavage,observe the intestinal microflora changes and intestinal morphological changes,to analyze the expression of intestinal tight junction protein by immunohistochemistry,and the detection of liver in the presence of the pathogen in the blood,explore the pathogenic bacteria through consumption of intestinal mucus as carbon source,thus breaking the intestinal barrier into the blood? Or the pathogen will gut barrier damage,which caused a large number of other bacteria into the blood into the liver,and finally into the gallbladder caused by cholecystitis? Method:1.by filling the stomach will from the bile of patients with acute cholecystitis separation of pathogenic bacteria were inoculated into a guinea pig in vivo,its effect on guinea pig intestinal morphology was observed by pathological section.And by specific primers for the detection of guinea pig serum in the presence of the pathogen,grinding fluid of liver tissue whether it contains the pathogenic bacteria,the immune fluorescence technique for the detection of intestinal mucosal epithelial cells and liver cells of tight junction proteins of the claudin 2 and closed occludin protein changes in expression level of to study whether the destruction of the guinea pig intestinal barrier function and hepatic barrier function.To explore the effect of the pathogenic bacteria on the biliary system2.using PCR-DGGE(DGGE,denaturing gradient gel electrophoresis)against the model animal fecal bacteria DNA with the qualitative and semi quantitative analysis,to detect intestinal bacteria group distribution of observed the changes of microbial community structure before and after the treatment.3.using real-time PCR to detect the level of mRNA in different treatment groups,intestinal mucus secretion related protein gene expression.Result:1.PCR-DGGE(DGGE,denaturing gradient gel electrophoresis).The results showed that antibiotic treatment for different days of intestinal bacteria group diversity showed a decreasing trend.2.Sequencing results showed that after the treatment of antibiotic,probiotic species decreased,pathogenic bacteria are added,which Bifidobacterium magnum(bifidobacteria)in the antibiotic processing after three days significantly reduced;Desulfotomaculum thermocisternum(genus Desulfotomaculum)amount of bacteria with antibiotics treatment days increase showed a decreasing trend;Prevotella oralis(oral Prevotella sp.)in antibiotic treatment is gradually disappearing;no Streptococcus constellatus,Streptococcus constellatus)in normal group,with antibiotic drug days increased,the bacteria gradually emerged.In addition to detect DesulFovibrio(Desulfovibrio desulfuricans),Hyphomonas neptunium(raw silk Aeromonas spp.),Tetragenococcus halophilus subsp.Halophilus(tetragenococcus halophilus).3.Real-time PCR results show pathogenic Escherichia coli group,intestinal mucus secretion protein MUC1 and Muc13,gene Muc15 expression level than that in the normal group and probiotic group increased significantly.4.E.coli o124 K72 infection in guinea pigs with experimental results shows that to E.coli group(G3)biopsy showed in the intestinal wall of large vacuolated tissues,goblet cell hyperplasia,and associated with mild inflammation.5.Immunohistochemistry showed that probiotics group(Anti+LGG)Claudin-2 content compared with the normal control group(CG)increased,but there was no statistical significance,and Claudin-2 in Anti+E.coli group than that in the normal group significantly increased(P < 0.05).The expression level of Occludin was just the opposite,but there was no significant difference between the groups.6.Each liver tissue grinding fluid were cultured and design of E.coli o124 K72 specially primers,specific amplification of bacteria liquid,but did not detect the pathogenic bacteria was found in liver tissue.Similarly in serum nor the pathogen,but probiotics group liver grinding fluid found the existence of a probiotic,Bacillus toyonensis.Conclusion:1.this topic by denaturing gradient gel electrophoresis(DGGE,denaturing gradient gel electrophoresis)against model animal fecal bacteria DNA with the qualitative and semi quantitative analysis,to detect the distribution of intestinal flora,before and after the treatment of flora structure changes were observed.Compared with the antibiotic treated and differences between normal intestinal flora composition,the system comprehensive reveal antibiotics to disruption of the intestinal flora to determine the successful modeling.2.real time PCR results show pathogenic Escherichia coli group,intestinal mucus secretion protein MUC1,Muc13,gene Muc15 expression level than that in the normal group and probiotic group increased significantly.Given that the gut's long suffering pathogen invasion,not only the structure will change,gene level will change and several of the mucus secreted proteins expression increased,also shows that intestinal inflammation of a symptom,a large number of secretion of mucus,intestine against external stimuli of a sense of self protection.3.Theimmunohistochemical staining results showed that E.coli(o124 K72)inoculated into a guinea pig in vivo,changes in the structure of intestinal mucosa,and upregulation of claudin 2 expression,down the expression of occludin intestinal barrier function and was related to the destruction.