| The first partEstablishment of reduced-size liver transplantation model in ratsObjective:The reduced-size liver transplantation model was established stabilityly in rat;To further volume reduction through rat liver regeneration after liver transplantation research provides important technical platform and prerequisite conditions.Methods:1.The donors were female and the receptors were male,which were healthy SD rats and weight range was260-280g, weight of the receptor was more than that of the donor,about 10g.2. Operation of donor was performed by only one person with the naked eye,during which reduced-size donor liver was performed. In taking in the process of donor liver resection left lateral lobe of the liver, triangle leaf and caudate lobe of the liver volume (about 40%-50%), The handle of self-made cuff was placed in the good front of portal vein and inferior vena cava respectively,which the tied ligature of pyloric veins was turned inside out of the self-made cuff,furthermore,the tied ligature was placed in the left of the self-made cuff;the same to inferior vena cava except that the tied ligature of right renal vein was placed in the right of the self-made cuff.Then the portal vein and inferior vena cava were received washing with self-made Perfusate respectively.3. Operation of the receptor was performed by two person with the naked eye,withimproved dual-cuff technique of Kamada and stay pipe of biliary traet.The fixed points of left and right were connected by anastomosis of 8"type turning inside out while inosculating inferior vena cava.4. The symptoms and signs、all complications、survival conditions were observed after liver transplantation modle and know the cause of death. Results:The operation time of gaining the donor and preparating the donor liver were 32±2minutes and 7±2minutes respectively.The operation time of gaining the receptor and the anhepatic were 44±3 minutes and 18±3 minutes respectively.And cold preservation time of liver donor was 51±3 minutes.The general successful rate was 92%,one-day survival rate was 95%、three-day survival rate was 85%^ five-day survival rate was 80% and seven-day survival rate was 75%.Conclusion:1.It was the better method of obtaining high quality and satisfied redueed-size liver in the rat that lobes of rat liver in operating on donor liver were removed after liver donor perfusion.The operation of donor and receptor was intercoordination,which may shorten cold preservation time of liver donor:The improved vascular anastomosis may shorten anhepatic time and reduce bleeding complications:It was worthy of being accepted model of redueed-size liver transplantation in the rat that the improved model may reduce in-operation and Post-operation complications after liver transplantation,and raise succeed rate of rat liver transplantation and survival rate of receptor.2. It was may be the foundation and Prerequisite for hepatic rebirth and immune experimental research after liver transplantation that improved model ofredueed-size liver transplantation in the rat was established sueeessfully.The second partThe function of NM23-E2 in liver cell regeneration after the reduced-size liver transplantation model in ratsObjective:Model of reduced-size liver transplantation in the rat was established sueeessfully.; Explore nm23-E2 gene affect the stage of the progress of liver regeneration after liver transplantation, through regulate the liver proliferation cycle of cyclinD1 and proliferating cell nucleus antigen (PCNA),it plays a positive role in promoting liver cells regeneration.Methods:In the first part model of reduced-size liver transplantation in the rat was established sueeessfully, In this experiment,five groups were designed, reduced-size liver transplantation (control,CON); NM23-E2 over-expression (transfected with NM23-E2 over-expression vector,OE);negative concorol of NM23-E2 siRNA (transfected with NM23-E2 siRNA negative concorol vector, siRNA-NC); negative concorol of NM23-E2 over-expression (transfected with NM23-E2 over-expression negative concorol vector,OE-NC);NM23-E2 siRNA (transfected with NM23-E2 siRNA vector,siRNA), Build, amplificate, sieve, accredit and package lentivirus vector of NM23-E2 which is over-expressed、interfered and negative concorol, inject the lentivirus vector before connect the portal vein,execute the rat five days after transfected,harvest the liver specimens and perform relevant tests, fluorescence detection of frozen section; Western-blot and immunohistochemical were used to analyze the expression levels of Cyclin D1 and NM23-E2 and PCNA protein; Q-PCR were used to analyze the mRNA expression levels of Cyclin D1 and NM23-E2 and PCNA.Results:Inject the lentivirus vector of NM23-E2 which is over-expressed and interfered before connect the portal vein, after five days,the corresponding fluorescent protein express obviously in liver tissue,it indicated that the lentivirus of NM23-E2 which is over-expressed and interfered had transfected into liver cells successfully.Both mRNA and protein expression of CyclinD1、NM23-E2 and PCNA of group OE were increased significantly (P<0.05) than that of group CON, on the other hand, Both mRNA and protein expression of Cyclin D1、NM23-E2 and PCNA of group siRNA were reduced significantly (P<0.05) than that of group CON.There were no difference in group CON、group OE-NC and group siRNA-NC (P>0.05), immunohistochemical showed that the positive expression rate of group OE was higher than that of group siRNA,and there was no difference in group CON、group OE-NC and group siRNA-NC.Conclusion:The lentivirus vector of NM23-E2 which is over-expressed and interfered had transfected into liver cells successfully.NM23-E2 over-expression vector play a positive regulatory role in promoting liver cells regeneration, and shRNA interference vector play a negative regulatory role in promoting livers cell regeneration. |
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