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1.Background and PurposeCirrhosis is a kind of chronic liverdiseases,which characterization is diffuse liver fibrosis,form false lobules and regenerate nodule.Cirrhosis is also common disease in China and one of the major causes of death.The basic performance is multiple system involvement clinically,with liver dysfunction and portal hypertension.Cirrhosis often accompany with intestinal infection,It was reported that the major infection source is the intestinal bacteria,especially Enterobacteriaceae.The study has researched the rela-tionship between cirrhosis and bacterial translocation.There are some difficulties in clinical study,such as collecting samples and patients often take antibiotic drugs in the treatment process.So we established the model of Bama minipig with Cirrhosis.Currently, the study on the relationship between gut microflora unbalance and infectious with the animal model of cirrhosis which induced by CCl4 has not been reported. The aim of my study is to explore the relationship between bacterial transloc-ation and Cirrhosis.We expected to construct a library of microbial strain of transferr-ed bacteria and then provid basic data for the further studying liver transplantation and investigating the correlation between the intestinal bacteria infection and bacterial translocation.2 Contents and results2.1 MethodsThe number of 16 Bama minipigs were 3 months old (provided by Department of Laboratory Animal Science,Third Military Medical University) were randomly divided into two groups:model group(6 male,4 female)and control group (5 male,1 female),all bama minipigs were not castrated. The model group was treated with 40% CCl4 olive oil solution by intraperitoneal injection.Injection volume was 0.25 ml/(kg.bw),once every four days. Cornmeal was their only food, free drinking 10% alcohol.The control group was fed by water and cornmeal only. 2.2 Detection of bacteriaLaparotomy and sample collections were taken under aseptic condition.Each of the abdominal organs was taken 0.5g, which was placed in 1 ml of PBS liquid,contain-ning liver, spleen, lung, kidney, and mesenteric lymph,and which was placed into a 2 ml tube,then take 100μl mixture liquid to inoculate onto brain heart infusion (BHI), then cultures 24-48h under 37℃We designed primers according to the 16S rRNA gene of variable region(V3 area) of bacterial, and then selected the same type of bacteria through PCR-DGGE.and then distinguished the DNA of transferred bacteria by polymerase chain reaction (PCR)and nucleotide sequencing. 2.3 ResultsSix Bama minipigs model of cirrhosis was induced successfully during the six months, success rate 60%, mortality rate 20%. The sequence result which in the light of GenBank(http://www.Ncbi.him.nih.Gov)shows that transferred bacteria mainly include Enterobacteriaceae,such as Enterobacter, Escherichia,Edwardsiella, followed by Gram-positive facultative anaerobic Staphylococcus, Corynebacterium, Enterococcus, Streptococcus, such as Staphylococcus aureus, Corynebacterium, Enterococcus faecalis, Streptococcus gallate solution, and other strictly anaerobic gram-negative bacteria. The number of bacterial translocation to the mesenteric lymph nodes is largest,8340CFU/g, followed by liver, kidney, the least is the lung,the normal control group without bacterial translocation.We construct a library of microb-ial strain of transferred bacteria.3.ConclusionsWe successfully obtained the animal model of liver cirrhosis, and proved that occured bacterial translocation during the formation of liver cirrhosis, and we successfully constructed a strain library of bacterial translocation. We identified translocating bacteria according to nucleic acid sequence of bacteria, the results show that the proportion of Gram-negative bacteria is 47.1%,Gram-positive bacteria is 58.3%,facultative anaerobic bacteria 83.3%,aerobic bacteria 16.7%, Escherichia coli 30.1%,Staphylococcus 25.0%, Corynebacterium 8.33%,Macrococcus 8.33% and so on.lt seems from the position of bacterial translocation,The incidence of bacterial translocation to blood, liver, spleen, lung, kidney and mesenteric lymph nodes was respectively 7.94%,12.98%,8.05%,7.68%,9.40%,53.95%.The study provides the basis reference for further researching clinical prevention and treatment of bacterial translocation.
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