The Construction Of Recombinant Adenovirus Vectors Ad-hIL-10 And Its Protective Effect On The Grafts Following Rat Orthotopic Liver Transplantation

Part 1:Establishment of the models of rat orthotopic liver transplantation and acute rejection of allograft in inbred ratsObjective:To establish the models of rat orthotopic liver transpantation(OLT)and acute rejection of allograft in inbred rats.Methods:120 cases of rat OLT were performed by modified Kamada's two-cuff technique in closed colony SD rats,followed by 30 cases of DA to Lewis OLT in inbred rat.The grades of acute rejection in allografts were judged according to Banff scheme.Results:Of the last 40 cases,the duration of anhepatic time was 17.98±3.03 min,and cold ischemia was 76.68±10.14 min.the successful rates of operation and 1w survive rates were 86.5%and 82.5%, respectively.Acute rejection was emerged on day 3 postoperation,the most typical pathological changes were found on day 7 postoperation.Conclusion:The rat OLT models were performed by modified Kamada's two-cuff technique.DA to Lewis rat strain combination is a stable acue rejection model.Part 2:The construction of recombinant adenovirus vectors Ad-hIL-10Objective:To procure recombinant adenovirus granule containing human interleukin 10 gene.Methods:The human IL-10 gene was acquired by PCR from pCDNA3.1-hIL-10 with a appropriate primer,the product of PCR were cloned into pShuttle-CMV by standard procedure.Recombinant pShuttle-CMV-hIL-10 and pAdEasy-1 were co-transformated into the BJ5183 cells to produce recombinant adenovirus plasmid(Ad-hIL-10).Then,the recombinant adenovirus plasmid were transfected in AD-293 cells to product packaging recombinant adenovirus granule.After identified,the desired recombinant adenovirus were purified by density gradient ultracentrifuge and titrated.Results:The pShuttle-hIL-10 was identified correct by sequencing and the recombinant adenoviral plasmid(Ad-hIL-10)containing target gene were selected for further study.Packaging recombinant adenovirus granule with high pure and titer(2×10¹⁰IU/ml)were generated after density gradient ultracentrifuge.Conclusion:Success in construction of recombinant adenovirus vectors and obtaintion packaging recombinant adenovirus granule with high pure and titer(2×10¹⁰IU/ml).Part 3:Effects of Ad-hIL-10 on preventing graft from cold ischemia reperfusion injury following rat orthotopic liver transplantationObjective:To study the mechanism and protective effects of Ad-hIL-10 on ischemia reperfusion injury(IRI)following rat orthotopic liver transplantationMethods:Male Sprague-Dawley rats were randomly divided as donors or recipients,rat orthotopic liver transplantation was performed after 12h preservation of graft in cold Lactated Ringer's solution. Ad-hIL-10 or control empty vector was deliver to the graft 48h before harvesting.4 groups were designed in the study,Group A:sham group; Group B:control group without vectors;Group C.control group with empty vector;Group D:Ad-hIL-10 group.Recipients were sacrificed 24h posttransplantation,serum levels of ALT and AST were measured,Part of the liver tissues were made into paraffin-embedded specimens to detect rat liver histological change and grade graft IRI(Suzuki's score);apoptosis were monitored by TUNEL;TNF-α,MIP-2,ICAM-1and hIL-10 mRNA of graft were measured by RT-PCR;Bcl-2,Bcl-xl,HO-1 and NF-κB gene products of graft were detected by Westem Blotting.Results:After treatment with Ad-hIL-10,mean value of all the assay tested was compared.The liver function(ALT and AST)in group D was significantly improved(P＜0.05),Suzuki's scores was significantly decreased(P＜0.05),level of hepatic TNF-α,MIP-2 and ICAM-1mRNA in group D was significantly decreased(P＜0.05),and the expression of hepatic Bcl-2,Bcl-xl,HO-1 gene products was significantly improved (P＜0.05),while NF-κB was decreased(P＜0.05).The grafts of group D revealed overexpression of hIL-10 mRNA,whereas,group A,B and C showed no expression of hIL-10 mRNA.Conclusion:(1)Doner pretreatment with Ad-hIL-10 led to the protective effects on IRI.Downregulated of proinflammatory cytokines TNF-α,MIP-2 and ICAM-1 mRNA and antiapoptotic effects may be associated with the protection against cold IRI.(2)hIL-10 protects against hepatic cold IRI,at least in part,by suppressing NF-κB activation and subsequent expression of proinflammatory mediators.(3)Ad-hIL-10 preventing grafts from apoptosis may be associated with upregulated expression antiapoptotic gene of Bcl-2 and Bcl-xl.(4)HO-1 as a downstream cytoprotective and antiapoptotic effector of hIL-10 in hepatic IRI after transplantation.Part 4:Effects of Ad-hIL-10 on preventing allograft from acute rejection following rat orthotopic liver transplantation.Objective:To study the mechanism and protective role of Ad-hIL-10 in acute rejection following rat orthotopic liver transplantationMethods:Orthotopic liver transplantation was performed in male inbred DA rats to Lewis rats.Ad-hIL-10,control empty vector or CsA was deliver to graft during the preservation.6 groups were designed in the study,Group A:sham group;Group B:received unmodified grafts;Group C:received hepatic grafts transduced empty vector;Group D: intramuscular injection 10mg/kg CsA at the time of transplantation;Group E:received hepatic grafts transduced Ad-hIL-10;Group F:Ad-hIL-10+ CsA(10mg/kg)group.All of groups were divide into two subgroups, Recipients of a subgroup were sacrificed on day 7 after transplantation, serum levels of ALT,AST and TBIL were measured,Part of the liver tissues were made into paraffin-embedded specimens to detect rat liver histological change and grade RAI(Banff scheme);apoptosis were monitored by TUNEL;the serum level of hIL-10 in group E and group F was tested by ELISA;IL-4,IL-2,TNF-γ,hIL-10 mRNA and hepatocyte-derived FasLmRNA of allograft were measured by RT-PCR; Bcl-2 and Bcl-xl gene products of allograft were detected by Western Blotting.Results:The allografts of group E and F revealed overexpression of hIL-10 mRNA,whereas,group A,B,C and D showed no expression of hIL-10 mRNA.Compared with group B,C and D,survival time of recipients in Group E and F was significantly prolonged(P＜0.05).After treatment with Ad-hIL-10,mean value of all the assay tested was compared,liver function(ALT,AST and TBIL)in group E and F was significantly improved(P＜0.05),RAI(according to Banff scheme)was significantly decreased(P＜0.05),level of hepatic IL-4 mRNA, hepatocyte-devived FasL mRNA and expression of hepatic Bcl-2,Bcl-xl gene products was significantly improved(P＜0.05).Compared with Group E,liver function(ALT,AST and TBIL)in group F was significantly improved(P＜0.05),survival time of recipients significantly prolonged(P＜0.05),RAI was significantly decreased(P＜0.05),level of serum hIL-10 and expression of hepatic hIL-10 mRNA was significantly improved(P＜0.05).Conclusion:(1)Doner pretreatment with Ad-hIL-10 preventing allografts from acute rejection in rat OLT.Subtherapeutic doses of CsA act synergistically with hIL-10 to significantly inhibit acute rejection of allograft.(2)Ad-hIL-10 preventing allografts from acute rejection may be associated upregulated the expression Th2 cytokine. (3)Ad-hIL-10 preventing allografts from apoptosis may be associated with upregulated expression antiapoptotic gene of Bcl-2 and Bcl-xl.(4)Doner pretreatment with Ad-hIL-10 preventing allografts from acute rejection may be associated with apoptosis of GICs by Fas-FasL approach.