The Construction Of RAAV-IκBαM And Its Effect Of On Orthotopic Liver Transplantation Model With Bile Ducts Ischemia-Reperfusion Injury In Rats

BACKGROUND:The incidence of biliary complications after liver transplantation is still high, and severely affects the survival rate and quality of life of liver transplantation patients. Prevention and treatment of biliary complications after liver transplantation are the key for improving the survival rate of recipient survival and long-term efficacy. Ischemia- reperfusion injury of bile duct is an important etiological factor in biliary complications after liver transplantation. Hepatic ischemia-reperfusion injury is the results of early activation of a variety of inflammatory factors. Transcription factor NF-κB plays an essential role in regulation of inflammatory response genes. Study the function of NF-κB in the course of ischemia-reperfusion injury and as interventions target probably has an exact protective effect on ischemia-reperfusion injury of bile duct after liver transplantation. The aim of our study was to investigate the protective mechnaism of the adeno-associated virus carrying IκBαM gene( rAAV- IκBαM) protect from the hepatic ischemia-reperfusion injury in rats.PartⅠEstablishment of orthotopic autologous liver transplantation model with bile ducts ischemia-reperfusion injury in rats and the activation of NF-κBObjective: To establish a stable orthotopic autologous liver transplantation model with bile ducts ischemia-reperfusion injury in rats, observe the surgical complications, survival rates and the activation of NF-κB in hepatic ischemia-reperfusion injury.Methods: Establishing orthotopic autologous liver transplantation model with bile ducts ischemia-reperfusion injury using the portal vein and abdominal aorta double perfusion technique. Weistar rats were randomly divided into two groups: liver transplantation-IRI group (LT-IRI group) with bile ducts ischemia-reperfusion injury surgical operation; Sham group (SH group) with only laparotomy and the surgical anatomy of hilar. Liver tissue and peripheral blood were collected at intervals of 2h, 24h, 72h and 7d after operation, the samples were used for immunohistochemical assay of NF-κB and Detection of enzymatic indicators of liver.Results: We carried out 80 cases of orthotopic autologous liver transplantation model with bile ducts ischemia-reperfusion injury, one of 60 cases of stable models with approximately 90% of surgical survival and 81.7% of week survival rates. Immunohistochemistry showed that after 2h LT-IRI group of NF-κB activated cells significantly more than SH group(p<0.01); while the liver function damage index (peripheral blood ALT) at each time point than those of LT-IRI group is also a marked increase in SH groups, with most significant at 24h postoperative comparing the two groups with significant difference(417.2±45.3 vs 57.6±5.4, p<0.01).Conclusion: Successfully establish the orthotopic autologous liver transplantation model with bile ducts ischemia-reperfusion injury with approximately 14±3 min anhepatic phase. There is NF-κB activation in the early stage of ischemia-reperfusion injury process.PartⅡThe construction and identification of recombinant adeno-associated virus vector carring IκBαM geneObjective: Construction of NF-κB super-portable IκBαM inhibitor gene recombinant adenovirus vector.Methods: Applying endonuclease BamHI and XholI double digestion the plasmid and PcDNA3.0-IκBαM, construction of pAAV-MCS-IκBαM with T4 ligase after plastic recycling and purification. Virus was packaged in 293 cells after digestion and sequencing, then substantial amplification of recombinant adenovirus vector and determination of virus titer with enrichment and purification. Results: We successfully constructed the recombinant plasmid pAAV-MCS-IκBαM with double digestion and conduction. We obtain the size of 6.4Kb and 970bp, respectively, for the vector and target gene fragment with double digestion of pAAV-MCS-IκBαM. The size of the 970bp fragment IκBαM PCR has been identified by PCR then identification of the correct sequencing. Determination of virus titer the separation enrichment and purification of recombinant adeno-associated virus titer was about 7×10~8pfu/ml～5×10~9pfu/ml .Conclusion: Successfully construction the recombinant adeno-associated virus vector carring IκBαM gene, titer of virus concentration and purification meet the experimental requirements.PartⅢThe protective effect of rAAV-IκBαM on orthotopic liver transplantation model with bile ducts ischemia-reperfusion injury in ratsObjective: To observe the expression of NF-κB and inflammatory molecules in downstream after rAAV-IκBαM transfected into the liver, investigate the apoptosis of bile duct endothelial cells and the changes of electron microscopy morphology. Discuss the effect of rAAV-IκBαM on orthotopic liver transplantation model with bile ducts ischemia-reperfusion injury in rats.Methods: Weistar rats were divided into three groups:GroupⅠfor the sham-operated group (only laparotomy), GroupⅡfor control group (only with liver transplantation-IRI surgical operation), GroupⅢfor control group rAAV-IκBαM transfection group (transfected with rAAV-IκBαM two days before surgery). Specimens were collected at intervals of 2h, 24h, 72h and 7d after operation, with conventional HE staining of extrahepatic bile duct and intrahepatic bile duct histology change,western blot detection of NF-κB p65 expression, RT-PCR determination of extrahepatic bile duct in liver tissue and liver tissue TNF-α, ICAM-1 mRNA expression, immunohistochemical detection of TNF-α, ICAM-1 expression, serum levels of TNF-αwith ELESA , the GGT changes in liver enzymes and bile duct endothelial cells apoptosis and morphological changes of electron microscopy.Results: rAAV-IκBαM transfection group compared with the control group: The expression of NF-κB p65 has a significant difference 2h after operation, nuclear protein levels were significantly lower, the nuclear NF-κB p65 protein was not obvious difference 24h after operation; The expression of TNF-αand ICAM-1 mRNA level and serum levels of TNF-αhas a significant difference 24h after operation; Immunohistochemistry showed the expression of TNF-αand ICAM-1 has a significant difference 24h,72h after operation; Indicators of liver damage (ALT,GGT) at various time points with significant differences, particularly in the 24h, 72h postoperative.The apoptosis of bile duct endothelial cells significantly reduced in the 24h, 72h after operation. And the morphological bile duct endothelial cells changes of electron microscopy significantly reduced in the 24h after operation.The transplant group and the sham operation group has significantly differences.Conclusion: The gene of IκBαM reduces the rat liver transplantation bile duct ischemia-reperfusion injury by inhibitting NF-κB nuclear translocation and its downstream gene expressing.