| Background and ObjectivesLiver transplantation is considered as the most effective treatment for end-stage liver disease.The biliary complications caused by ischemia reperfusion injury(IRI)of bile duct which is one of the main reasons for the long-term effect of liver graft.Consequently,the strategies to attenuate IRI would considerably improve the quality of life and survival of the transplant recipients.The heme oxygenase-1(HO-1),a stress-responsive enzyme is recognized as catalyzing the rate-limiting step in the principal degradative mechanism of heme,which is highly inducible.More attentions have been paid to HO-1 because of its cytoprotective,antioxidant,main-taining microcirculation,modulating the cell cycle and anti-inflammatory functions.However,whether the overexpression of HO-1 can be induced to reduce the IRI of biliary tract,and liver transplantation is transplant donor to the recipient,it is still unclear whether the donor or recipient can play a main protective to the graft.In this study,the model of cold ischemia reperfusion injury in orthotopic liver transplantation was established.We constructed a recombinant adenovirus expression vector encoding rat HO-1 genes and targeting shRNA-HO-1 recombinant adenovirus vector,and transfected the two recombinant adenovirus into the donor or receptor rat liver respectively and the expression of HO-lwas induced or silenced in rats.To observe the effect of HO-1 on bile secretion and transportation,and to analyze the internal relationship between HO-1 and bile duct epithelial cell damage,cholangitis and the fibrosis process in bile duct epithelium.By changing the donor and/or receptor HO-1 expression to observe its effect on biliary ischemia reperfusion injury,and to provide new ideas and methods for the clinical improvement of ITBL after liver transplantation.Methods1.To construct Adv-HO-1 and Adv-HO-1-siRNA,and to transfect them into rats respectively.After 24h,to observe the expression level of HO-1 protein of liver was detected by Western-blot.2.The establishment of a model of rat orthotopic liver transplantation:To establish a reliable animal model of rat orthotopic liver transplantation by using the two-cuffed technique,and to observe the complications of perioperative and livability within 24 hours after liver transplantation.Compared the morphological changes of liver tissue between normal group and operated group,and to confirm the success of construct the model of ischemia reperfusion in rat liver transplantation.3.In vivo studies of rats:320 SD rats were equally and randomly divided into 5 groups,which were group A receiving injection of blank adenovirus(adv),group B with donor receiving Adv-HO-1 and recipient receiving blank adenovirus,group C with donor and recipient both receiving Adv-HO-1,group D with donor receiving Adv-HO-1-siRNA and recipient receiving blank adenovirus,and group E with donor and recipient both receiving Adv-HO-1-siRNA at 24 h before liver transplantation by the rat via tail vein(lml,3x1O8pfu/ml).On dl,d3,d7 and d14,serum and liver was isolated for analysis of liver function(ALT?AST?GGT?ALP?TBIL).Inflammatory cell infiltration by HE staining,and ultrastructure of liver by transmission electron microscopy.Immunohistochemical analysis of inflammatory cells infiltration into bile duct The expression of HO-1,Bsep,Mrp2 and Ntcp by western blot as well as the expression of the fluorescence intensity of Laminine(red)and CK18(green)protein determined by double-staining essay.Results1.To successfully construct Adv-HO-1 and Adv-HO-1-siRNA,and the amplification sequence was proved correct by sequencing.The protein expression of HO-1 in donor liver after adenovirus transfection before transplantation was measured by western blot and showed significantly higher HO-1 expression in Adv-HO-1 group compared with that in blank group and Adv-HO-siRNA group(P<0.05).Moreover,the expression of HO-1 in Adv-HO-1-siRNA group was significantly inhibited compared with that in blank group(P<0.05).2.Results of establishment of a model of rat orthotopic liver transplantation:To establish a reliable animal model of rat orthotopic liver transplantation.175 cases of rat orthotopic liver transplantation were performed,12 cases of them died within 24 hours after surgery.There is no statistical difference between the groups(P>0.05),160 cases of them were survival more than 24 hours,the 24 hours survival rate is 93%.The morphological changes of the operation group were consistent with ischemia reperfusion injury,and the model was established successfully.3.Results of recombinant adenovirus transfected in vivo:?Hepatic enzyme:Hepatic enzyme as represented as the level of ALT,AST,TBIL,ALP and GGT,was evaluated on d1,d3,d7 and d14 after reperfusion.Levels of ALT,AST,GGT,ALP and TBIL were all significantly reduced in group B and C compared with those in group A(P<0.05).However,compared with group A,group D and E showed significantly higher levels of ALT,AST,GGT,ALP and TBIL(P<0.05).?Immunohistochemistry:Bile duct inflammation was assessed by immunohistochemistry staining of CD3,CD45R.Ggroup E showed increased numbers of CD3+ and CD45R+ T lymphocytes in portal tract compared with group A on dl after reperfusion with few inflammatory cells infiltration in group C than group A.On d7 after transplantation,bile duct epithelial cells necrosis,detachment and proliferation were observed in group E as demonstrated by Ki67 staining,whereas,all these pathological changes were not obvious in group C.?Histopathological changes in microscopy:The changes of liver pathology were evaluated by HE staining.Hepatocytes degeneration,focal point necrosis,edema in portal tract with inflammatory cells infiltration were observed in group A,B and C.Liver from group D and E displayed obviously abnormal structure of hepatic lobule,hepatocytes degeneration,necrosis and proliferation with infiltration of several inflammatory cells.? Liver ultrastructure:The ultrastructure of liver was evaluated by transmission electron microscopy on d14 after reperfusion,compared with group A,obviously swelling and deformation of bile duct columnar epithelium,obstruction of bile duct,disappearance of microvilli,mitochondrial swelling,matrix cavitation as well as endoplasmic reticulum expansion in group D and E,while group B and C which displayed less injury of epithelium and normal structure of mitochondria.?Confocal microscopy analysis of double immunofluorescence staining LN and CK18:Confocal microscopy was performed to evaluate the changes of hepatocytes and bile duct after transplantation on d14 using fluorescence-labelled anti-LN and CK18 antibody.Compared with group B and C,group D and E showed heavier injury of bile duct,abnormal structure of bile duct epithelium with continuity of basement membrane and alignment of hepatocytes around bile duct.?Western blot:total proteins were isolated from liver for analysis of the expression of HO-1 Mrp-2,Bsep and Ntcp by western blot using GADPH as control.Expressions of HO-1,Mrp-2,Bsep and Ntcp in group B and C were significantly higher than those in group A,D and E with more obvious increase on d7(P<0.05).Conclusions1.To successfully construct and transfect the Adv-HO-1 and Adv-HO-siRNA into the donor rat.2.To successfully establish a convenient?economic and reliable animal model of rat orthotopic liver transplantation by reformed "two cuffed technique " and restricted hepatic artery by "arterial stent".3.Our study demonstrated that overexpression of HO-1 can ameliorate the damage of bile duct and liver.4.Give priority to the donor,the higher expression of the donor HO-1 was more significant than the higher expression of the receptor HO-1 to ameliorate the damage of bile duct and liver,it might be a new therapeutic target in the treatment of ITBL after liver transplantation. |
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