The Research Of MiRNA-122 In The Evaluation Of The Quality Of DCD Liver In Pigs

BackgroundLiver transplantation is the most effective treatment for end-stage liver disease.But,the the contradiction between liver shortage and patients waiting for liver transplantation is still severe.Some country began to use cardiac death donors in1960 s,but replaced by brain death donor later.Until the 1980 s,transplant centers in Europe and the United States renewed attention cardiac death donors.Compared with the DBD,DCD donor is inevitable to experience hypotension,shock,hypoxia ischemia injury in cardiac arrest process.Long time warm ischemia injury may lead to primary transplantation liver function、hepatic artery thrombosis、 hepatic ischemic biliary lesions and serious complications.So,the quality evaluation of DCD liver is particularly important,in order to avoid giving up available donor liver and using not suitable donor liver resulting in serious adverse consequences.Currently,the quality assessment methods for DCD liver are cold-warm ischemia time,degree of liver fatty liver,blood sodium level,age,hepatitis infection,tumor and other aspects.There is not an objective index to reflect the degree of liver injury and prognosis.Therefore,looking for a sensitive and objective evaluation index for DCD liver is a serious problem.miRNA-122 is a member of the miRNA family,accounts for about 70% of total miRNA in liver cells and undetectable in other tissues.In the embryonic period,miRNA-122 levels in liver cells began to increase,and the content of each cell can reach 66000 copies in adult rat liver.In human liver cells can reach 135000 copies.The sequences of miRNA-122 are highly conserved from human to zebrafish,and situ hybridization in zebrafish also shows that liver specificity of miRNA-122 is also highly conserved.miRNA-122 involved in development and differentiation,gene expression regulation and metabolism of liver.Study shows that nonalcoholic fatty liver disease,viral hepatitis,hepatocellular carcinoma,drug-induced liver injury were significantly correlated with miRNA-122.With the the research of miRNA-122 in liver transplantation recently,some studies indicated that miRNA-122 can evaluate the warm ischemia injury during liver hypothermia mechanical perfusion.Experiments also show that miRNA-122 can predict early acute rejection reaction and ischemic biliary complications after surgery.But,the research is rarely reported in the correlation between expression level of miRNA-122 and the level of liver injury in the DCD process and the liver function recovery prediction after surgery.Objective : The purpose of this study was to research the relationship between miRNA-122 level of DCD liver tissue and liver injury and postoperative recovery of the liver function.Further mechanism research of miRNA-122 change in hypoxia damage to the donor liver tissue was taken.In order to provide a new evaluation index for DCD liver injury and new therapeutic target for the protection of DCD liver.Methods:1.Set up the DCD liver transplantation model in pig.Obtain fresh liver tissue and liver tissue for pathological at different warm ischemia time(0 minutes,10 minutes,20 minutes,30 minutes).2.Establish hypoxemia DCD liver transplantation model in pig.Obtain fresh liver tissue and pathological required liver tissue at different hypoxia treatment time(15 minutes,30 minutes,60 minutes)and after hypoxia with warm ischemia.3.Extract total mi-RNA in liver tissue.Measure the micro RNA-122 expression levels in liver tissue at different warm ischemia time and different hypoxia treatment time by q RT-PCR method.4.Multi sample variance analysis method was used to assess whether monitoring indicators expression had statistical difference with liver hypoxia and warm ischemia time.The histogram showed the indicators for monitoring trends over time.Results: 1.In the pig DCD model,liver histopathology was not changed much,but the level of miRNA-122 had increased after 10 minutes of warm ischemia.2.Donor liver pathology change and miRNA-122 expression levels had gradually increased with the extension of the warm ischemia time.miRNA-122 levels in the tissue gradually increased at different warm ischemia group(10 minutes,20 minutes,30 minutes)with statistical difference(P < 0.05).3.Histopathology had little change after hypoxia treated for 15 minutes,30 minutes,60 minutes for liver.But,miRNA-122 expression level obviously increase after hypoxia treated 60 minutes and had statistically significant difference(P < 0.05)with hypoxia treated group for 15 minutes,30 minutes.4.In hypoxia combined with warm ischemia group,the expression level of miRNA-122 had significant difference among different hypoxia treated time(p<0.05).Conclusions: 1.The “asphyxia” method has the advantages of relatively safe and controllable to establish a porcine DCD transplantation model.It can well simulate the liver transplantation process of clinical Maastricht III.2.miRNA-122 expression levels began to increase at warm ischemia 10 min,and continued to rise with ischemic time extension.miRNA-122 levels can reflect the degree of ischemic damage can be as an evaluation index for warm ischemia injury.3.miRNA-122 expression level obviously increase after hypoxia treated 60 minutes.miRNA-122 levels can reflect hypoxia injury to the donor liver.4.miRNA-122 level had good evaluation function on hypoxia combined with warm ischemia injury of donor liver.