A Study On The Intestinal Molecular Flora And Serumal Metabonomic Spectrum In The Model Of Liver Transplant Rat With Hepatic Cirrhosis

Orthotopic liver transplantation(OLT) is to date the best and most durable treatment for patients with end-stage liver disease such as acute or chronic hepatic failure and cirrhosis.For various improvements have been made in both surgical and therapeutic techniques,the successful rate of liver transplantation increased and the survive time became longer.All complications are still major causes of post-transplant mortality.The infection is the second cause next to the acute rejection.The incidence or mortality is 47%- 80%or 13%- 36%respectively.The post-transplant infection not only lead to rise the duration and fee of hospitalization,but also it adds the high risk of graft liver dysfunction.The most often pathogen infected liver transplantation include the bacteria(48%),fungi(22%) and virus(12%).The Endogenous infection often occurred in 30 days after liver transplantation.About 31%of liver transplant patients with at least one time caused by multiple drug-resistant infections.The 69%of bacterial infections in post liver transplantation are caused by multi-drug resistant bacteria. Because of the anatomical structures and functions in close contact between liver and intestinal tract,intestine as the body's largest reservoir of bacteria and endotoxin,it can release a variety of toxic substances under certain conditions,which will bring the occult cause of endogenous infection.Gut microflora imbalances,intestinal barrier damage and increased intestinal permeability,immune dysfunction are the major reasons for infection of liver transplantation.This study focused on the gut microflora and serum metabolic changes before and after liver transplantation.Aimed at finding the changes in gut microflora of liver transplantation infection,as well as searching specific metabolic target related liver transplantation.It can provide of new ideas and new ways for future exploration through the reconstruction,to optimize gut microflora,to reduce the incidence of infection after liver transplantation,and to promote long-term health of transplant recipients survive.In this study,there are three distinctive characteristics.Firstly,we chose the liver transplantation model of rat with liver cirrhosis induced by carbon tetrachloride. Secondly,we discovered the changes of the bacterial composition and serum metabolism of dynamic range by using 16S rDNA molecules microecological technology and H~1 NMR.It was different from the traditional bacteria culture.There were no similar reports up to now.Research Methods1.The rat liver cirrhosis model induced by Carbon tetrachloride (CCL4)Class clean Sprague-Dawley(SD) rats with 50,were equally divided between male and female,weight 180g-200g.Dividing into two groups:control group(10) and model group(40).Model preparation methods:the control group of rats with sterile peanut oil solution subcutaneously;model in rats by subcutaneous injection in sterile 40%solution of carbon tetrachloride in peanut oil.After administration of 4 weeks,8 weeks,12 weeks,16 weeks,the two were put to death in model rats,observing the progress of liver pathology,continuous 16-week rat model of liver disease.10 Models and 5 control group were executed at the time of the first 16 weeks.Last,the remaining 22 liver cirrhosis in rats and 5 control rat liver transplantation standby.2.Liver transplantation model after cirrhotic ratChoose a clean class,weight 250g - 300g Sprague-Dawley(SD) male rats for 19 as the donor.Receptor:CCL4-induced SD rat model of liver cirrhosis 14 and SD rats in control group 5.Divided into three groups:the same gene liver transplantation group(9 rats),the control group(5 rats),sham operation group(liver cirrhosis in rats,it is only open abdominal suture,5 rats).The classic two Kamada Sleeve law was applied in the orthotopic liver transplantation model in rats. 3.Specimen collection(1) Blood and feces:all the blood and feces were collected CCL4 before delivery, liver cirrhosis,the seven days and 30 days after liver transplantation.(2) The liver cirrhosis model 10 and 5of the control group were killed when the cirrhosis model became successful;and the 19 mouse receptors were put to death 30 days post-transplant treatment.Remove the blood cavity,liver,spleen and kidney.4.Detection methods(1)~1H-NMR for detection of serum metabolites.(2) Liver,Spleen and Kidney immediately to do aerobic and anaerobic grinding culture,biochemical identification of bacterial types,analysis of the incidence of translocation.(3) Extraction from the faeces of total DNA,using specific primers for the case of Enterococci,Enterobacter, Bacteroides,Clostridium,Bifidobacterium,Lactobacillus quantitative fluorescent PCR; on the levels of intestinal bacteria to change six clear of bacteria make further 16S DNA PCR-DGGE,analysing of different groups of bacteria molecular structure of ecological patterns and differences in the strains,using cloning and sequencing analysis.5.Data analysis methodsCategorical variables and measurement data were compared using the chi-square and T test respectively.MATLAB software was used to do principal component analysis(PCA).Results1.There is significant difference in the rate of bacterial translocation to liver, spleen,kidney between liver cirrhosis group(10%) and liver transplantation group(66%) (P<0.05).The liver was the most common site of bacterial translocation,followed by the spleen and the kidney.The often translocation types of bacteria were Enterococcus faecalis,Pseudomonas aeruginosa and Escherichia Coli in the liver transplantation group,while Pseudomonas aeruginosa dominated in liver cirrhosis group.2.We observed the changes of six major kinds of fecal bacteria at five time points (before CCL4 administration,4 weeks after administration,liver cirrhosis,7 days and 30 days after the liver transplant) by using the real-time fluorescence quantitative PCR. The results showed that the quantities of Bacteroides genus,Clostridium and Enterobacter spp were deccreasing in liver cirrhosis group comparing with the control group,However,they gradually increased in liver transplantation group and reached normal level in one month after liver transplantation group.Bifidobacterium increased in the process of liver cirrhosis and began to decrease significantly 7 days after liver transplantation,but it had partial recovery after 30 days.The changes of Lactic acid bacteria and enterococci were not obvious in liver cirrhosis process.3.We analysed the intestinal molecular flora structure at the 4 time point including CCL4 before delivery,liver cirrhosis,7 and 30 days after liver transplantation by using PCR-DGGE method based on 16S rDNA V3 area.The results showed that there was small difference of intestinal molecular flora structure between individuals in the control group.In the contrary,there was significant difference in the liver cirrhosis group.Moreover,the intestinal molecular flora structure significantly changed in the group of 30 days after liver transplantation comparing with the control group. Interestingly,the smallest difference was seen in the groups of 7 and 30 days post transplantation groups.4.Application of group-specific PCR-DGGE technology,further analysis of intestinal predominant bacteria such as Clostridium genera,the genus Bifidobacterium and Bacteroides.The study found that:(1) The intestinal bacteria molecular structure had small individual differences in Clostridium genera group,the genus Bifidobacterium group and Bacteroides,group CCL4 before delivery.(2) Compared to the before CCL4 administration group,the fundamental changes of Bacteroides structure have taken place in liver cirrhosis group and 30 days after the liver transplant group.Of which 30 days after transplant was the farthest away from the normal group But it had posed a similar bacteria spectrum between liver cirrhosis groups and the seven days after transplant groups.There were 16 species of Bacteroide to have largest contributions to the divide the groups.Furthermore,most of them could not be cultured. (3) The Clostridium molecular ecology according to the PCA indicated:individual distribution of dispersion and large individual differences inner the liver cirrhosis group and 30 days group.The Clostridium composition before cirrhosis was obviously different from after cirrhosis,Furthermore,the difference between groups was greater than the inter-group differences.Clostridium basic ecological structure of the molecule returned to the normal levels 7 days after operation.However,Clostridium bacteria composition after 30 days transplantion was totally different from the normal group,and there was the farthest distance between the two groups.There were 12 species of Clostridium to have largest contributions to the divide the groups.Moreover,most of them could not be cultured.(4) Bifidobacterium DGGE fingerprinting was carried out principal component analysis,the structure of the composition of Bifidobacterium relatively simple,Even though the intestinal Bifidobacterium molecular structure had small individual differences and formed as cluster CCL4 before delivery.But at the time of liver cirrhosis,it should not be clustered,distributing in all quadrants.It can collect together when 7 days and 30 days after liver transplantation again,which showed that the composition of Bifidobacterium can restore in acute phase rejection,and then keep stability for at least 30 days.5.The dynamic changes law of the serum metabolic spectrum(18 substances) were explored in total of 22 rats before and after liver cirrhosis or liver transplantation using the ~1H NMR combined with multivariate statistical analysis.The results showed that: lactic acid,high-density lipoprotein,unsaturated fatty acid,betaine increased significantly,and the branched-chain amino acids such as glutamate,glutamine,valine, etc;O-acetyl glycoprotein,N-acetyl-glycoprotein,glucose;low-density lipoprotein and very low density lipoprotein,fatty acid;acid decreased when Cirrhosis formed.With the implantation of liver,the abnormal metabolites gradually restored,but the metabolite recovery time varies.Further analysis by the use of PCA:The metabolic spectrum was completely separated in cirrhosis model group,the normal control group and 7 days after liver transplantation group.They distributed in different regions,showed that their metabolism in a completely different composition.But there is some overlap in the metabolism spectrum between 30 days after liver transplantation and normal group. This showed that there is a part of the restoration of metabolic spectrum at this time. ConclusionsIn this study,the intestinal molecular flora and serum metabonomic spectrum were carried out in different times of liver cirrhosis and liver transplantation,using by systemic biology method.It discovered that the different categories were in the different disease status under the six major intestinal bacteria and spectrum of metabolic changes. At the same time,it was confirmed that the existence of an imbalance of intestinal flora in the liver transplantation rat.It will help to restore the structure of normal flora,to protect the intestinal mucosal barrier,to reduce bacterial translocation and transplant infections by using the probiotics(including probiotics,etc.)...