Rat Small Volume Liver Transplantation Ischemia-reperfusion Experimental Study, The Mechanism Of Injury And Adenovirus-mediated Cardiotrophin-1 Transfected With Their Protection

Partâ… The establishment of small-for-size liver transplantation model in rats and the primary study of its characteristics and mechanisms of graft injury in early stage after ischemia and reperfusionObjectiveTo establish a practical and stable small-for-size liver transplantation animal model in rats and investigate the possible injury mechanisms in early stage after ischemia and reperfusion by comparing the injurious degree of small-for-size and full-size liver transplantation.Our studies provide the the theoretical and experimental basis for exploring new therapeutic strategies in small-for-size liver graftsMethodsMale Lewis rats were divided into three groups randomly:Groupâ… , sham group;Groupâ…¡,100%graft size liver transplantation group(whole size group);Groupâ…¢,40%small-for-size liver transplantation group. 40%small-for-size liver transplantation model was performed according to kamada's two-cuff method and performing hepatectomy in vitro after skilled operation training.Survival rate and early graft function(AST and ALT levels)were observed in different groups;morphological changes were examined by light and electron microscope;the content of malondialdehyde(MDA)in liver grafts after 6 hours was detected; enzyme-linked immunosorbent assay(ELISA method)for TNF-αLevels in Liver Grafts.Additionally,we used myeloperoxidase(MPO)activity assay to determine neutrophil infiltration into the grafts at 6 h after reperfusion;apoptotic cells in liver grafts were detected by TUNEL assay.Results1.Compared with whole liver group,the survival rate was significantly decreased in small liver graft group(P＜0.05).Seven-day survival rate was significantly decreased from 83.3%(10/12)in whole liver group and 100%(10/10)in sham group to 33.3%(4/12)in small liver group.2.Serum AST and ALT levels were markedly increased after reperfusion in two graft groups.The AST and ALT levels at 2,6 and 24 hours after reperfusion in small liver graft group were significantly higher than those in whole liver grafts(p＜0.05).3.HE staining showed disruption of lobular architecture,apparent hepatocelluar degeneration accompanied by focal necrosis;similarly, electron microscopy also showed severe mitochondria swelling accompanied by an irregular large gap between sinusoidal endothelial cells and loss of microvilli in 40%small-for-size liver graft.While in whole graft group,minimal damage were observed at 6 hours after reperfusion.4.The content of MDA after 6 hours was increased significantly in small-for-size(1.83±0.32)and whole size liver grafts(1.19±0.23) compared with sham group;especially in small-for-size group (p＜0.01).5.Hepatic TNF-αlevels and MPO activity at 6 hours after reperfusion were increased significantly in small-for-size and whole size liver grafts compared with sham group;especially in small-for-size group, 40%small-for-size group vs whole graft group,TNF-α(7.91±1.07 vs 4.56±0.73 p＜0.01);MPO(6.79±0.74 vs 4.15±0.62 p＜0.01).6.TUNEL staining showed that the number of apoptotic hepatocytes was increased significantly in small-for-size liver graft(20.4±4.3) compared with those in whole size liver graft(11.2±2.1,p＜0.05).ConclusionsPractical and reliable animal model of small-for-size liver transplantation in rats was established successfully,grafts underwent severe reperfusion damages in the rats with small-for-size liver grafts compared with whole size liver grafts;more severe reactive oxygen species,accelerated acute inflammatory responses and hepatocelluar apoptosis plays an important role in graft injury of small-for-size liver transplantation.Partâ…¡Construction and identification of murine cardiotrophin-1 gene recombinant adenoviral vector,viral production,amplification and purification ObjectiveTo construct and identify recombinant adenovirus vector Ad-CT-1 containing mouse CT-1 cDNA,and to produce the virus containing mCT-1,using adenoviral packaging cell line 293.MethodsMurine cDNA encoding CT-1 gene was cloned across the SalI -NotI sites of plasmid PGEX4T-3-CT-1 and then into the pAdTrack-CMV shuttle vector via same sites.The pAdTrack-CMV vector encodes EGFP under the control of a separate cytomegalovirus(CMV)promoter to act as a marker for transfection.The plasmid was linearized by restriction digestion with PmeI and cotransformed into E.coli BJ5183 cells with pAdEasy-1.Recombinants were selected for kanamycin resistance and confirmed by restriction analysis.The linearized recombinant plasmid was transfected into an adenovirus packaging cell line AD293,using Lipofectamine in T-25 flasks according to the manufacturer's instructions. Transfection and viral productions were monitored by GFP expression. The crude viral stock was further amplified and purified by CsC1 banding. The concentrations of AdCT-1 and AdEGFP were determined by plaque-forming assay,and expressed as plaque-forming units(pfu).ResultsThe recombinant adenovirus vector Ad-CT-1 were digested with restriction analysis,agarose gel electrophoresis and PCR assays results showed it contained mCT-1 DNA fragment,The DNA sequencing of CT-1 was identical to the report in Genebank and did not reveal any mutation.The titer of AdCT-1 was very higher to 10¹²pfu/ml.ConclusionsOur data revealed that the recombinant adenoviral vector containing the mouse CT-1 cDNA have been constructed successfully,the AdCT-1 and the AdEGFP viral have higher titers,which will provide material basis for the following study on ischemia and reperfusion in small-for-size liver transplantation.Partâ…¢Adenoviral Cardiotrophin-1 transfer improves survival and early graft function after ischemia reperfusion in rat small-for-size liver transplantation modelObjectiveThis study was to investigate whether donor liver Adenoviral cardiotrophin-1 gene transfer ameliorates ischemia reperfusion injury in rat small-for-size liver transplantation model and its possible mechanismsMethodsSyngeneic male Lewis rats were used as donors and recipients,the experiments were conducted in three groups of rats:(â…°)AdCT-1 group,(â…±) AdEGFP control group,and(â…²)saline control group.AdCT-1 and AdEGFP vectors were diluted to 3×10⁹ pfu/ml with 1 ml saline for intravenous injection to donor rats.AdCT-1,AdEGFP or 1 ml 0.9%saline was injected via the penile vein into prospective donor animals.Four days later,the donor liver was harvested.A rat nonarterialized orthotopic 40% small-for-size liver transplantation model without veno-venous bypass was performed according to the techniques previously described in partâ… , liver function(serum AST and ALT levels)at 2,6 and 24 hours after reperfusion were examined;Survival rate of three groups was investigated;Morphological changes at 6 and 24 hours of liver grafts were examined by light and electron microscopy;the apoptosis was detected by TUNEL assay;cell survival signal proteins(Akt and phospho-Akt;ERK and phospho-ERK;Stat-3 and phospho-Stat-3),antiapoptotic protein bcl-2 and pro- apoptotic protein cleaved caspase-3 were assessed by westernblotting.Results1.Donor liver adenoviral CT-1 gene transfer leads to CT-1 overexpression in post-transplanted small-for-size grafts.2.Adenoviral CT-1 gene transfer improved animal survival and early liver function of small-for-size grafts.The differences in survival rates between the three groups were significant,and the survival rate of the AdCT-1 group was improved compared to the AdEGFP and saline control groups.The 7-day survival rate was significantly improved from 33.3%(5/15)in the saline control group and 40%(6/15)in AdEGFP control group to 73.3%(11/15)in the AdCT-1 group(P＜0.05);serum levels of AST and ALT were significantly lower in the AdCT-1 group compared to the saline and AdEGFP control groups at 2, 6,and 24h after reperfusion(p＜0.05 or＜0.01).There were no differences between saline and AdEGFP control group.3.In the saline and AdEGFP groups,H-E staining showed apparent hepatocellular degeneration accompanied by scattered necrotic cells at 6h after reperfusion;severe disruption of lobular architecture,more severe hepatocellular degeneration accompanied with extensive necrosis was present at 24h after reperfusion.Whereas minimal damage was observed in the AdCT-1 treatment group.Likewise,in the saline and AdEGFP groups,hepatic ultrastructural changes noted by electron microscopy included mitochondria swelling accompanied by an irregular large gap of sinusoidal endothelial cells,loss of microvilli at 6h after reperfusion,an disruption of the endothelial cell integrity at 24h after reperfusion.While in AdCT-1 group,both the cell nucleus and cellular organelles have no significant breakdown.4.TUNEL staining showed that the frequency of apoptotic cells was diminished in AdCT-1 group;the apoptotic index,calculated as the percentage of TUNEL-positive nuclei divided by the counter- stained nuclei,was significantly diminished in the AdCT-1 group compared with saline and AdEGFP control groups(p＜0.01),no differences were observed between the two control groups.5.Westernblot assay revealed that no significant differences in total Akt, extracellular-regulated kinase(ERK)and Signal transducer and activator of transcription-3(Stat-3)were observed between the three groups.However,p-Akt,p-ERK and p-Stat-3 levels were significantly up-regulated in the AdCT-1 group compared to the saline and AdEGFP groups(p＜0.05 or p＜0.01).6.Westernblot assay also demonstrated that anti-apoptotic bcl-2 levels were significantly up-regulated and pro-apoptotic cleaved caspase-3 was markedly down-regulated in the AdCT-1 group compared to the saline and AdEGFP groups(p＜0.01).ConclusionsDonor liver adenoviral cardiotrophin-1 gene transfer improves survival and reduces graft injury after ischemia and reperfusion in small-for-size liver grafts,the protective effect of cardiotrophin-1 on small-for-size graft injury is mediated,at least in part,by activation of Akt,ERK and stat-3 survival signaling pathways.In addition,involved in enhanced anti-apoptotic protein bcl-2 expression and diminished pro-apoptotic molecule caspase3 activation.