Analysis Of Bacterial Diversity Of Intestinal Microbiota In Liver Transplant Rats And Human After Hepatic Cirrhosis By PCR-DGGE And Rep-PCR

ObjectivesLiver transplantation (LT) is the most effective treatment for patients with end-stage liver disease such as acute or chronic hepatic failure and cirrhosis. Infection and rejection is still the most common complications after LT. Gut and liver are closely associated not only in the anatomy, but also in the function. As one of the largest reservoir of bacteria and endoxin, intestine can release a variety of toxic substances, leading to enterogenic infections. Previous studies have showed that bacterial translocation was obviously found in patients with cirrhosis, while Gram-negative aerobic bacteria increased significantly, mainly Enterobacter, and anaerobic bacteria such as Bifidobacterium and Lactobacillus decreased. However, the changes of bacterial diversity of intestinal microbiota in LT recipients with cirrhosis have not been explored deeply. The dysbiosis of gut microecosystem, loss of gut barrier function, increased intestinal permeability, immunity dysfunction, surgical ischemia-reperfusion injury and so on, were the important causes of infection after LT. In present study, we investigated the bacterial diversity of intestinal microbiota in LT rats and human with hepatic cirrhosis by PCR-DGGE, genus-specific Real-time PCR and rep-PCR, exploring the changes of bacterial diversity of intestinal microbiota after LT. Materials and Methods1. Firstly, we established CCL4-induced cirrhosis model of SD rats, choosed healthy SD rats as the donor, then transplanted for SD rats of cirrhosis model. We collected stool specimens of 4 time point incluing normal control group, cirrhosis model group,7d and 30 d group after LT, and then stored in -80℃within 1 hour.2. Collecting stool specimens of 10 healthy subjects,23 patients of cirrhosis,9 LT patients from 4 months to 4 years, and then stored in -80℃within 1 hour.3. Choosing the QIAamp DNA Stool Mini Kit combined with bead-beating, a widely confirmed methods for extracting the total genomic DNA from feces which stored in -80℃C, and then universal bacterial primers for the V3 region of 16S rRNA genes were used to amplify the extracted bacterial genomic DNA, and the PCR products were used for denaturing gradient gel electrophoresis(DGGE).4. Bacterial genus-specific primers were used to analyze the changes of the 6 predominnant bacteria in the gut microbiota.5. Five rep-PCR primers (BOX-PCR, ERIC-PCR, ERIC2-PCR, (GTG)5-PCR, REP-PCR) were used to amplify the bacterial genomic DNA which extracted from feces of rats and human, and then compared with the PCR-DGGE fingerprinting.Results1. The CCL4-induced cirrhosis models of SD rats were established, and LTs were operated successfully. One rat died after LT and rejection was mild.2. The bacterial composition and structure of intestinal microbiota in rats could be divided into three clusters by PCR-DGGE distinctly, which the patterns were similar in human. And we found that there were more bacterial richness in hepatic cirrhosis group and LT groups. Intestinal microbiota, such as predominant bacteria-Bacteroides, decreased in liver cirrhosis group, while the other minority bacteria increased. Bacterial diversity in 7d after LT began to recover, but not yet restore to normal level after 30d.3. Our real-time PCR results have showed that the genera of Bacteroides, Clostridium, Bifdobacterium and Lactobacillus were significantly decreased in liver cirrhosis group in rats, while the the genera of Enterobacteriaceae and Enterococcus were significantly increased. In human being, the genera of Clostridium and Bifdobacterium were significantly decreased in liver cirrhosis group, while the the genus of Enterococcus was significantly increased.4. Among the five rep-PCR genomic fingerprinting methods, ERIC-PCR, ERIC2-PCR and REP-PCR could also discriminate each other according to the molecular fingerprinting.5. PCR-DGGE fingerprinting primary analyzed the diversity of predominant intestinal mcirobiota, and rep-PCR genomic fingerprinting technology researched the overall changes. Both of the two methods show similar power of discrimination of the bacterial diversity of intestinal microbiota, and the resolution of PCR-DGGE fingerprinting is better.ConclusionThe diversity of gut microbiota in liver transplant SD rats and human after hepatic cirrhosis was changed significantly, especially in liver cirrhosis group, while the gut microbiota tended to restore after LT. Both of the two molecular fingerprinting methods (rep-PCR and PCR-DGGE techniques) can be applied to analyze the dynamics of intestinal bacterial diversity in LT model with hepatic cirrhosis, which can provide an effective method to monitor the eubiosis or dysbiosis of the gut microbiota.