【作者】 戴小明；

【导师】 叶启发；

【作者基本信息】 中南大学 ， 外科学， 2007， 博士

【摘要】 第一部分大鼠肝移植模型改进目的:探索建立稳定大鼠原位肝移植模型的方法,为进一步研究大鼠肝脏冷保存再灌注损伤提供模型基础。方法:选用清洁级SD大鼠建立同种异体原位肝移植模型,手术采用“Kamada双袖套法”,并在其基础上做适当改进,如:采用静脉注射泵代替手工灌注,留取供体肝上下腔静脉膈环以上2mm静脉进行成形,并于静脉壁左右侧分别预吊缝线等。手术分为第一阶段预实验（50次）和第二阶段正式实验（50次）进行,在第二阶段又按术式分为“Kamada双袖套法”（26次）和“改良Kamada双袖套法”（24次）,并比较二种术式建立大鼠肝移植模型的效果。结果:预试验期间手术存活率为34.00%（17/50）,正式实验手术存活率达90.20%（46/50）。“改良Kamada双袖套法”的供、受体手术时间,袖套准备及供肝修整时间,无肝期都比“Kamada双袖套法”有明显缩短;术后一周的存活率显示“Kamada双袖套法”为65.21%（15/23）,“改良Kamada双袖套法”为95.73%（22/23）,统计分析具有显著性差异（P＜0.05）。结论:1.“改良Kamada双袖套法”具有无肝期和手术时间短,手术成功率和术后存活率高的优点,是建立大鼠原位肝移植模型较为理想的术式;2.本实验通过对取肝、修肝、受体肝脏切除、肝上下腔静脉吻合、围手术期处理等几方面的改进,提高了模型的稳定性和成功率。第二部分匹立尼酸对不同冷保存时间大鼠肝脏内PPARα、NF-κB和iNOS的影响目的:探索匹立尼酸对冷保存大鼠肝脏内PPARα、NF-κB和iNOS的影响,研究其对移植肝冷保存损伤的作用和作用机制。方法:将60只SD大鼠随机分成二大组:匹立尼酸预处理组（P₂）,30只,于术前2d、1d、1h分别经尾静脉一次注入10%DSMO8ml/kg.d+Pirinixic acid 3mg/kg.d;对照组（C₂）,30只,则注入相应剂量的10%DSMO溶剂。按“改良Kamada双袖套法”切取大鼠供肝,然后,每组又按在4℃林格氏液中冷保存时间不同分为五个亚组:冷保存Oh组,6只;冷保存2h组,6只;冷保存4h组,6只;冷保存6h组,6只;冷保存8h组,6只。分别于上述不同时间点分批次采取肝中叶标本,应用逆转录聚合酶链反应（RT-PCR）、蛋白质印迹技术（Western-Blot）检测过氧化物酶体增殖物激活剂受体（PPAR）α、诱导型一氧化氮合酶（iNOS）mRNA及表达蛋白量的变化,免疫组织化学法检测肝细胞内核因子（NF）-κB活性变化,并比较二组的病理学特征。结果:在对照组（C₂）和匹立尼酸预处理组（P₂）肝组织中,随着冷保存时间的延长PPARαmRNA和表达蛋白的量都逐渐降低,但同一时间点P₂组比C₂组要高,统计学比较具有显著性差异（P＜0.05）。在C₂组和P₂组肝组织中iNOS mRNA和表达蛋白的量随着冷保存时间延长都升高,但同一时间点P₂组比C₂组要低,统计学比较也具有显著性差异（P＜0.05）。同一冷保存时间点匹立尼酸预处理组肝细胞内NF-κB的激活水平低于对照组,病理损伤也较对照组轻。结论:1、PPARα在冷保存的大鼠肝脏中表达下调,并随冷保存时间的延长表达水平逐渐下降（在林格氏液中冷保存,8小时内）;2、Wy-14643对大鼠肝脏冷保存损伤具有保护作用,其保护作用是通过改善PPARα的表达下调、抑制NF-κB的活化和iNOS的病理性高表达来实现的。第三部分匹立尼酸对大鼠移植肝冷保存再灌注损伤的保护作用和作用机制目的:进一步挢索匹立尼酸对大鼠移植肝冷保存后再灌注期损伤的作用,并了解其作用机制。方法:将90只清洁级SD大鼠随机分成三组:对照组（C₃组）,18只,于术前2d、1d、1h分别经尾静脉一次注入10%DSMO 8ml/kg.d,只行开关腹及肝脏游离手术;匹立尼酸处理组（P₃组）,36只;模型组（M₃）,36只。M₃和P₃组动物再随机分成供体、受体,P₃组供体于术前2d、1d、1h分别经尾静脉一次注入10%DSMO 8ml/kg.d+Pirinixicacid 3mg/kg.d,M₃组供体则注入相应剂量的10%DSMO溶剂,后二组按前述的“改良kamada双袖套法”建立肝移植模型。然后,三组动物又以术后再灌注时间点不同再分为三个亚组:即T₁:再灌注2小时,T₂:再灌注6小时;T₃:再灌注24小时;各6只。分别按上述时间点检测肝组织中PPARα、iNOS mRNA及表达蛋白的量、髓过氧化物酶活力（MPO）、超氧化物酶活力（SOD）、丙二醛含量（MDA）、肝细胞中NF-κB活性和血清中谷丙氨酸转氨酶（ALT）、门冬氨酸转氨酶（AST）、肿瘤坏死因子（TNF）α、巨噬细胞炎性蛋白（MIP）-2的变化,并比较三组肝脏的病理学差异。结果:对照组（C₃）术后不同时间点肝组织中PPARα、iNOS mRNA及表达蛋白的量、MPO活力、SOD活力、MDA含量、肝细胞中NF-κB活性和血清中ALT、AST、TNFα、MIP-2的都无明显变化,肝脏的病理学无显著改变。模型组（M₃）和匹立尼酸预处理组（P₃）PPARαmRNA及表达蛋白的量、SOD活力、随着再灌注时间的延长而逐渐下降,6h达到最低点,以后逐渐恢复,同一时间点三组的统计学比较,具有显著性差异（P＜0.05）,P₃比M₃组下降的幅度低,统计学分析也具有显著性差异（P＜0.05）。相反,肝脏中iNOS mRNA及表达蛋白的量、MDA含量和血清中ALT、AST、MIP-2在M₃和P₃组则于再灌注开始时上升,6小时达到高峰,以后下降,同一时间点三组的统计学比较,具有显著性差异（P＜0.05）,P₃组比M₃组升高的幅度低,统计学分析也具有显著性差异（P＜0.05）,但MIP-2在P₃组和M₃组却无显著性差异。M₃和P₃组肝细胞中的NF-κB活性、血清中TNFα随着再灌注时间的延长于开始时上升,2小时达到高峰,以后逐渐下降,同一时间点三组的统计学比较,具有显著性差异（P＜0.05）,P₃组比M₃组低,统计学分析也具有显著性差异（P＜0.05）,但血清中TNFα在P₃组和M₃组却无显著性差异。肝脏病理学检查,P₃组比M₃组损伤轻。结论:1、在冷保存-再灌注损伤早期（再灌注后6h内）,大鼠移植内PPARα表达继续下调,于再灌注6h达到最低点,24h时有所回升;2、Wy-14643对大鼠移植肝前期（再灌注后24h内）冷保存-再灌注损伤具有保护作用,其作用是通过改善PPARα的下调表达,抑制肝细胞内NF-κB的激活,调控肝脏内一氧化氮合酶（iNOS）的转录来抑制iNOS源性NO的生成,进而抑制iNOS源性超氧化物和过氧亚硝基阴离子的损害,抑制中性粒细胞的聚齐,并且能够提高肝细胞自身的抗氧化能力来实现保护作用。更多还原

【Abstract】 PartⅠThe 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.PartⅡThe 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.PartⅢ: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.更多还原