| Partâ… The improvement in the establishment of orthotopic liver transplantation model in ratsObjective:To investigate the surgical procedures and establish stable model of liver transplantation in rats in order to study the cold preservation reperfusion injury of graft.Methods:The models of orthotopic liver transplantation were made on SD rats."Two-cuff technique described by Kamada[TCTDK]" was used with some modifications.Such as:the vena injection pump was used to perfuse liver through abdominal aorta and portal vein instead of handiwork perfusion,the 2mm suprahepatic inferior vena cava above diaphragm annulation was kept to shape,and two sides of which were sutured with suture line before anastomosis of suprahepatic inferior vena cava,et al.Two phases were included in this experiment:preliminary experiment(50 times)and formal experiment(50 times).The effects on the establishment of liver transplantation model were compared between TCTDK(26 times)and "Two-cuff technique described by Kamada with some modifications[TCTDKM]"(24 times)in the formal experiment.Results:The operation survival rate was 34%(17/50)in the preliminary experiment,and 90.20%(46/50)in the formal experiment. The times of donor operation,recipient operation,preparation of cuff, graft trimmed and anhepatic phase were shorten significantly by the TCTDKM than the TCTDK.The survival rate was 65.21%(15/23)for TCTDK,and 95.73%(22/23)for TCTDKM.There was significant difference between two groups(P＜0.05).Conclusion:1.The TCTDKM is a good technique to liver transplantation in rats,with the advantage of shorter time in operation and anhepatic phase,higher operation survival rate and survival rate at 1 week after operation than others.2.The stability and achievement ratio of liver transplantation model in rats were increased by the improvement of the techniques on the graft obtained and trimmed,the anastomosis of suprahepatic vena cava and resection of liver in the recipient,and the treatment in ambi-operation phase in our experiment.Partâ…¡The influence of pirinixic acid on the peroxisome proliferators-activated receptors-a(PPARa),iNOS,NF-κB in cold preservation liver of rat.Objective:To investigate the influence of pirinixic acid on the peroxisome proliferators-activated receptors-a(PPARa),iNOS in the hepatic tissue,and NF-κB in hepatic cells of rat at different cold preservation time,and to study the effect of pirinixic acid on hepatic cold preservation injury in rats,and its mechanism.Methods:60 SD rats were randomly divided into control group(C2) and pretreated group(P2);rats in P2 group were pretreated with 10% DSMO 8ml/kg.d+Pirinixic acid 3mg/kg.d injected intranvenously by vena caudalis on 2d,1d and 1h before operation,30 rats;and rats in C2 group were did in the same quantity of 10%DSMO at the same time,30 rats.Grafts were obtained by TCTDKM.Then,the two groups were divided into five subgroups respectively with the different cold preservation time in 4℃Ringer's solution,including:Oh cold preservation group,6 rats;2h cold preservation group,6 rats;4h cold preservation group,6 rats;6h cold preservation group,6 rats;8h cold preservation group,6 rats.The sample of median lobe of rat liver was obtained in batch at the above-mentioned time point to detect the level of mRNA of PPARa and iNOS by reverse transcription-polymerase chain reaction (RT-PCR),the quantity of expression protein of PPARαand iNOS by Western-blot,and the activity of nuclear factor-κB(NF-κB)in hepatic cells by immunohitochemical method.Pathological characteristics of the cold preservation liver were compared between two groups.Results:With the time of cold preservation prolonged,the levels of mRNA and the quantities of expression protein of PPARa were decreased in C2 group and P2 group,and which in P2 group were higher than in C2 group at the each same cold preservation time(P＜0.05).On the other hand,with the time of cold preservation prolonged,the levels of mRNA and the quantities of expression protein of iNOS were increased in C2 group and P2 group,and which in P2 group were lower than in C2 group at the each same cold preservation time(P＜0.05).The activity of NF-κB in hepatocyes of cold preservation liver was lower in P2 group than in C2 group at the each same cold preservation time.There were slighter pathological injuries in P2 group than in C2 group.Conclusion:1.The downregulation expression of PPARαand expression of PPARαdecreased with the development of the time of cold preservation in cold preservation livers of rats(within 8h,in 4℃Ringer's solution)2.There was protective effect of Wy-14643 on the hepatic cold preservation injury in rats,which was implemented to protect liver from injury by improving the downregulation of PPARα,and suppressing the activity of NF-κB and high pathological expression of iNOS.Partâ…¢:The protective effect and mechanism of pirinixic acid on cold preservation-reperfusion injury of transplanted liver in ratsObjective:To investigate the effect of pirinixic acid on cold preservation-reperfusion injury of transplanted liver in rats,and its mechanism.Methods:90 SD rats were randomly divided into three groups, control group(C3):18 rats,only treated with abdomen dissection and suture,and liver dissociation;model group(M3):36 rats;and pretreated group(P3):36 rats.Rats in M3 and P3 group were randomly divided into donors and recipients respectively;donors in P3 group were pretreated with 10%DSMO 8ml/kg.d+pirinixic acid 3mg/kg.d injected intranvenously by vena caudalis on 2d,1d and 1h before operation.Donors in M3 and rats in C3 group were did in the same quantity of 10%DSMO at the same time before operation.The liver tansplantation models were established by TCTDKM.Then,three groups were divided into three subgroups respectively with the different reperfusion time after operation, Including:T1:2h after reperfusion group;T2:6h after reperfusion group; T3:24h after reperfusion group;6 rats in each group.Samples of right and median lobe of liver were obtained in batch to detect the mRNA levels of PPARαand iNOS,the quantities of expression protein of PPARαand iNOS,the activity of MPO,the contents of MDA in the transplanted liver, and the activity of nuclear factor-κB(NF-κB)in hepatic cells at above-mentioned time point,and AST,ALT,TNFαand MIP-2 in serum were also measured.Pathological characterics of the liver were compared in three groups.Results:There were no significant change on the levels of mRNA and quantities of expression protein of PPARαand iNOS,activity of MPO and SOD,contents of MDA in livers,activity of NF-κB in hepatic cells,the levels of AST,ALT,TNFαand MIP-2 in serum,and the pathological characteristic observation in C3 group at each time point. Levels of mRNA and quantifies of expression protein of PPARα,and the activity of SOD were decreased in M3 and P3 group as the time of reperfusion time prolonged,which reached the lowest point at 6h after reperfusion,and then increased after that time.There were significant differences at the each same time after reperfusion among the three groups(P＜0.05),there was lower decrease in P3 group than in M3 group, and also significant difference between P3 group and M3 group.On the other hand,levels ofmRNA and quantities of expression protein of iNOS, and the activity of MPO and contents of MDA in livers,and the levels of MIP-2 in serum were increased as the time of reperfusion time prolonged in M3 and P3 group,which reached the highest point at 6h after reperfusion,and then decreased after that time.there were significant differences at the each same time point after reperfusion among the three groups(P＜0.05),there was lower decrease in P3 group than in M3 group, which was the level of mRNA and the quantity of expression protein of iNOS,and the activity of MPO,the content of MDA in liver.Significant difference between P3 group and M3 group was also found,but not for levels of MIP-2 in serum between P3 group and M3 group.The activity of NF-κB in hepatic cells and levels of TNFαin serum were increased in M3 group and P3 group in the first 2 hours after reperfusion,reached the highest point at 2h after reperfusion,and then decreased alter that time. There were significant differences at the each same time point alter reperfusion among the three groups(P＜0.05).There were lower increase of the activity of NF-κB in hepatic cells in P3 group than in M3 group,and also significant difference was found between P3 group and M3 group,but not for levels of TNFαin serum between P3 group and M3 group.There were slighter pathological injuries in P3 group than in M3 group.Conclusion:1.In the first 6 hours after reperfusion,the downregulation expression of PPARa and expression of PPARa decreased with the development of the time of reperfusion in the transplanted liver of rats, which reached the lowest point at 6h after reperfusion,and then recovered after that time;2.Wy-14643 protected the transplanted liver from cold preservationreperfusion injury in rats by improving the downregulation of PPARα, suppressing the activity of NF-κB,and controling the transcription of iNOS in hepatic cells,and thus inhibiting the damage generated by superoxide and peroxynitrosonegion that derived from iNOS,suppressing the accumulation of neutrophil,and elevating the ability of antioxygen of hepatic cells within 24 hours after reperfusion. |
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