| Background:IRI (ischemia-reperfusion injury) is inescapable to liver transplantation, and may bring grafts on primary nonfunction which is one of the most important factors of retransplantation. So the study on alleviating IRI conduces to shrink the gap between organs available for transplantation and the number of patients awaiting an organ. Meanwhile, the gene therapy is one of the clinic ways to be practiced rapidly at recent years. It is an alluring therapeutic approach to utilize the technique of transforming functional genes into organ grafts to alleviate IRI.Part one:An Animal Model of ROLT by Modifying Kamada's MethodObjective: to establish an animal model of ROLT by modifying Kamada's method.Methods: 120 cases of ROLT were performed by ameliorating the surgical technique based on Kamada's method and improving the treatment at around-operation period, and were investigated the operation time of donors and recipients, anhepatic period and survival time.Results: In contrast with the initial stage, there were significant differences from the operation time, the livability of 24 hours and one week and the incidence of the operation complication.Conclusion: It can establish an ideal animal model of ROLT via improving the surgical technique based on Kamada's method and performing a certain amount of cases. Part two:Construction of Recombinant Adenovirus of Human Heme Oxygenase-1 GeneObjective: To construct the recombinant adenovirus of human heme oxygenase-1 gene.Methods: (1) The segment of hHO-1 gene was liberated from the cloning vector of pOTB7-hHO-1 by restriction endomuclease EcoRI and Bg1II; (2) the segment of hHO-1 gene was ligated into adenovirus shuttle vector pSGCMV after digested with EcoRI and Bg1II by standard procedure, and the production pSGCMV- hHO-1 was identified and selected. (3) pSGCMV- hHO-1 and backbone vector pBHGloxP△E1E3 were cotransformed into the adenoviral packaging 293 cells by the Cre-mediated sit-specific recombination method, and it was named Ad-HO-1. (4) After the security of Ad-HO-1 was evaluated by VT001/VT002, his infectional efficiency in vitro was also tested.Results: (1) the positive recombination pSGCMV- hHO-1 was obtained after hHO-1 gene was ligated into pSGCMV, and the hHO-1 gene of the verified pSGCMV- hHO-1 was correct. (2) Ad-HO-1 which was obtained by the pathway of the Cre-mediated sit-specific recombination method was in high titer which reached 2.0×1010pfu. (3) The obtained Ad-HO-1 did not contain wide-type adenovirus, and could be transformed effectively in vitro.Conclusion: Ad-HO-1 which can express effectively and safely in vitro can be obtained by the Cre-mediated sit-specific recombination method.Part three:hHO-1 gene transfer protects rat grafts from IRI followed OLTObjective: To investigate how recombinant adenovirus carrying HO-1 which was constructed by ourselves alleviate hepatic IRI after transplantation.Methods: Two groups of SD rats (n=8) were studied. Control donors were intraperitoneally pretreated with Ad-EGFP (2.5×109pfu) 36 hours before their livers were harvested; and experimental donors were intraperitoneally pretreated with Ad-HO-1(2.5×109pfu) 36 hours before their livers were harvested. 6 hours after reperfusion, serum ALT, AST and LDH were measured, and apoptotic cells by TUNEL and the apoptotic ratio by Flow Cytometer were analyzed; and the expression of HO-1 and antiapoptotic(Bcl-2 and Bcl-xl) and proapototic(caspase-3) gene products were determined by Western-blot. The positive cells of HO-1, TNF-α, caspase-3 and macrophage were stained by immunohistochemistry. The ultrastructure of grafts was observed by electronic microscope.Results: (1) The expression level of HO-1 in the experimental group was significantly higher than one in the control. Whereas the liver function indicator ALT, AST and LDH and the apoptotic ratio of hepatic cell decreased significantly. (2) By Western-blot,the expression of antiapoptotic(Bcl-2 and Bcl-xl) gene product in the experimental group was increased significantly, and the expression of proapoptotic(casepase-3) gene product was weakened obviously. (3)By immunohistochemistry ,as compared with the control group,in the experimental group a lot of the positive cells of HO-1 were expressed and mainly lay at the vascular area,and there were neither massive macrophages infiltration nor caspase-3 and TNF-αoverexpression. And there were more apoptotic cells around the Disse space in the control group than in the experimental group by TUNEL(.4)By electronic microscope,there were a few classic apoptotic cells to be observed in the control group,but the ultrastructure of the experimental group was almost normal.Conclusion: (1)The recombinant Ad-HO-1 can be intraperitoneally transfered into donor liver and be expressed obviously in vascular area of grafts. (2) HO-1 can alleviate ischemia-reperfusion injury in rat liver by suppressing apoptosis and inflammation which relates to significantly modulating proapoptotic (caspase-3) and antiapoptotic (Bcl-2 and Bcl-xl) pathways and depressing macrophage infiltration and TNF-αexpression. |
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