Experimental Study On HO-1 To Prevent Liver Allograft From Damage After Transplantation

Objectives: To establish a rat model of stable orthtopic livertransplantation(OLT)using cuff technique, the establishment of a rat OLTis the precondition to perform such study.Methods: OLT in 200 SD rats which received Wistar liver wereestablished by using "the two-cuff technique" with some modification incuff tube, recipients anesthesia abdominal aorta perfusion and theanastomosis of suprahepatic vena cava, portal vein, infrahepatic venacava.Results:The time of vascular anastomosis for suprahepatic venacava was 10 to 15 minutes, the anhepatic phase was 12 to 18 minutes,more than 75ï¼…of the grafted animals survived for seven days. Bleeding,thrombosis, infection and biliary obstruction Objective: To construct and identify HO-1 gene recombinantreplication-deficient adenovirus(Ad-HO-1).Methods and Results: The construction and identification of HO-1gene recombinant replication deficient adenovirus.In this study, we usedAdEasy system to construct HO-1 recombinant replication-deficientadenovirus. Firstly, the whole HO-1 gene was obtained from the PRHO-1plasmid via Xhoâ… +Hindâ…¢digestion, and inserted into Pucl8 plasmid.Then HO-1 gene was digested from Puc 18-HO-1 by Kpnâ… +Hindâ…¢andsubcloned into shuttle vector of pAdTrack-CMV. The resulting plasmidpAdTrack-CMV-HO-1 was linearized with Pmeâ… and cotransformedinto BJ5183 cells together with adenovirus genomic plasmid ofpAdEasy-1. The pAdEasy-1 was E1 and E3 deleted and its E1 functioncould be complemented in 293 cells. Recombinants were selected withkanamycin and screened by restriction enzyme analysis. The recombinantadenoviral construct was then cleaved with PacI to expose its ITR(Inverted Terminal Repeats) and transfected 293 cells to produce viralparticles. Recombinant adenoviruse was purified by Double CesiumChloride Gradient and titered (infectious particles, IP) as TCID50 (tissueculture infectious dose 50).Adenovirus stocks were tested for the absence of replication-competent adenoviruses by PCR amplification of the E1adenoviral region. The resulting recombinant adenovirus was identifiedby PCR of HO-1 gene and expression of HO-1 protein by western blot.Conclusion: HO-1 gene recombinant replication-deficientadenovirus was successfully constructed.2.HO-1 gene recombinantreplication-deficient adenovirus had the qualities of high efficiency, stableexpression of HO-1 and safety in cultured tubular epithelial cells. Objective: To investigate the protected effects of HO-1 genetransfection on liver allografts based on rat allograft model.Methods and Results: we established inbred rat livertransplantation model and infected liver allograft with recombinantHO-1/Ad. Then we observed the survival time, HO-1 expression andpathological changes in liver allograft, and measured the changes ofhepatic function. The results indicate that HO-1 expression is correlatedwith the changes of hepatic function, delayed lymphocytes infiltration,decreased number of lymphocytes infiltrated into liver aUograft and mildpathological changes in liver allograft. There is no expression oftransfected HO-1 gene in the other organs all the time.Conclusion: HO-1 can protect liver allograft from graft rejectionand prolong the survival time of liver alloRraft, so it has a therapeuticpotential in graft rejection.