【作者】 张莹；

【导师】 别平；

【作者基本信息】 第三军医大学 ， 外科学， 2006， 博士

【摘要】 目的:一、研究冷保存—再灌注肝移植大鼠肝细胞线粒体DNA ATPase6、ATPase8基因表达变化以及对其编码的F₀F₁-ATPase表达和功能的影响,从转录、翻译水平探讨冷保存—再灌注后ATPase6、ATPase8基因在线粒体有氧呼吸功能改变中的作用,揭示冷保存—再灌注损害中能量代谢障碍的分子机制。二、研究线粒体DNA上游调节基因NFR-1和mtTFA对冷保存肝移植大鼠肝细胞线粒体DNA ATPase6、ATPase8基因表达的影响,探讨NFR-1和mtTFA对冷保存肝移植大鼠肝细胞线粒体DNA调节作用及其机理。方法:1.Wistar大鼠186只,采用改良“二袖套法”制作大鼠肝移植模型,动物随机分为A组（冷保存30min组）,B组（冷保存6 h组）,C组（冷保存12 h组）和D组（假手术对照组）,分别于制模后于12h、24h、3d、5d、7d采集标本,保证每时相点存活大鼠6只。2.观察各组大鼠肝脏的病理改变、肝功能、肝组织SOD、ATPase、ATP含量及线粒体形态。3.采用RT-PCR方法检测mtDNA ATPase6、ATPase8 mRNA和NFR-1和mt TFAmRNA的表达变化。采用Western-blot法检测肝细胞线粒体F₀F₁-ATPase蛋白的表达变化。结果:1.各组手术时间及无肝期基本接近,无统计学差异,肝脏复流后全肝灌注均匀,动物并发症较少。冷保存30min、6h、12h三个时相点肝移植大鼠的损伤程度存在明显差异。2.C组再灌注后肝脏病理变化、肝功能损害、肝组织SOD、ATPase、ATP含量降低较A组、B组显著（P＜0.05）。再灌注7d后B组、C组肝功能趋于正常,但C组肝组织SOD、ATPase、ATP含量仍增高。3.冷保存再灌注后早期（12h）,A、B、C三组ATPase6、ATPase8 mRNA表达均降低,C组显著低于A组、B组（P＜0.05）,并且ATPase6、ATPase8 mRNA量的变化趋势一致,F₀F₁-ATPase表达量也降低（P＜0.05）,再灌注24h后A组、B组、C组ATPase6、ATPase8 mRNA表达开始升高,F₀F₁-ATPase表达量也增加,C组ATPase6、ATPase8 mRNA表达以及F₀F₁-ATPase表达量的升高较A组、B组缓慢。4.冷保存再灌注后早期（12h）,A、B、C三组mtTFA及NRF-1 mRNA表达降低,以C组最为显著（P＜0.05,与A组、B组相比）,24h后各组mtTFA及NRF-1 mRNA表达开始升高,ATPase6、ATPase8 mRNA表达也升高,并且升高趋势与mtTFA及NRF-1mRNA表达增高基本一致。结论:1.本动物模型缩短了肝移植大鼠的手术无肝期,提高了大鼠肝移植的成功率和存活率,具有良好的重复性和稳定性。并且冷保存30min、6h、12h三个时相点肝移植大鼠存活率及不同损伤程度可满足研究冷保存再灌注损伤及修复的要求。2.供肝冷保存—再灌注对肝细胞及其线粒体有明显的损害作用,表现为血清ALT、AST升高,肝组织ATPase、SOD降低、ATP生成减少,肝细胞凋亡明显,并随冷保存时间的延长而进一步加重。再灌注后,随着时间的延长SOD含量逐步升高,肝细胞及其线粒体病理损害逐渐减轻,ATP合成逐渐增加。提示在冷保存-再灌注过程中,肝细胞及其线粒体的损伤可能与氧自由基活动的增加有关。3.供肝在冷保存-再灌注后肝细胞ATPase6、ATPase8 mRNA表达异常,细胞能量供应急剧下降,并且与ATPase6、ATPase8 mRNA表达异常密切相关,表明线粒体功能障碍可能与线粒体编码ATPase6、ATPase8基因之间存在联系。4.冷保存-再灌注后ATPase6、ATPase8基因表达及其编码的线粒体呼吸链蛋白F₀F₁-ATPase蛋白表达量减少,并且冷保存时间的延长,再灌注后变化越显著,对ATP能量合成影响越大。同时ATPase6、ATPase8 mRNA量的变化趋势基本一致,提示ATPase6、ATPase8基因也可能表达共同的mRNA,mtATPase6、ATPase8基因改变后F₀F₁-ATPase蛋白表达异常,是导致能量合成障碍的主要原因。5.供肝在冷保存—再灌注后,mtTFA mRNA和NRF-1 mRNA变化与ATPase6、ATPase8 mRNA表达变化相一致,推测mtTFA和NRF-1可能通过基因转录调节ATPase6、ATPase8基因表达,改变线粒体呼吸链转运电子、合成ATP的能力。更多还原

【Abstract】 Backgroud:Liver transplantation is the only effective therapy for end-stage liver diseases,and has been carried out successfully all over the world.However,cold preservation-reperfusion remains one of the most important factors in determining the effect of liver transplantation. Cold preservation-reperfusion is closely related to primary nonfunction（PNF） and delayed function recovery of the graft.Our experiments aimed to study the changes and regulatory mechanisms of mt DNA encoding ATP6,8 of hepatocytes after liver transplantation in rats.Objective:（1） To study the changes o f the expression of mt DNA encoding ATP6,8 of hepatocytes in rat liver transplantation with cold preservation-refusion,and explore the relationship between the expression of F₀F₁-ATPase and function induced by changes of ATP6,8 genes. （2）To investigate the mechanism of the energy metabolism disorder of mitochondria and the cellular damages induced by cold preservation-refusion in hepatocytes.（3） To observe the effects of NRF-1 and mtTFA on the expression of ATP6,8 genes and study the molecular mechanism of NRF-1 and mtTFA on repair of mitochondria DNA injury.Methods:Orthotopic liver transplantations were performed in wistar rats using the cuff technique as described by Kamada with modifications.Rats were randomly divided into four groups namely:group A（cold preservation of 30 min）,group B（cold preservation of 6 hours）,group C（cold preservation of 12 hours）-and group D（sham operation）.Samples were collected at 12 h,24 h and on 3^st,5^th,7^th days after operation.The pathological changes of liver tissue,liver function,SOD,ATPase,ATP of liver and mitochondrial morphology were observed in each group.The changes of the expression of NFR-1,mtTFA and mtDNA encoding ATPase6,ATPase8 mRNA were determined by RT-PCR.The changes of the expression of F₀F₁-ATPase protein was examined by Western-blot. Results:（1）The pathological changes of liver tissue and liver function were more severe in group C than in groups A and B.SOD,ATPase,ATP of liver and mitochondrial morphology in group C were significantly lower than in groups A and B（p＜0.05）.The liver function gradually recovered in groups B and C,but SOD,ATPase and ATP of liver in group C were increased gradually on day 7 after operation.（2） The expression of ATPase6,ATPase8 mRNA were decreased in groups A,B and C on 12h after operation.ATPase6,ATPase8 mRNA were the lowest in group C when compared with groups A and B（p＜0.05）.The expression of ATPase6,ATPase8 mRNA increased after 24h in groups A,B and C.The changes of F₀F₁-ATPase protein expression were consistent with the expression of ATPase6,ATPase8 mRNA.Both ATPase6,8 mRNA and F₀F₁-ATPase protein in group C increased slowly compared with groups A and B after 24h.（3） The expression of mtTFA and NRF-1mRNA were decreased in groups A,B and C at 12h after operation,and mtTFA and NRF-1 mRNA were the lowest in group C（p＜0.05）.The expression of mtTFA and NRF-1 mRNA were consistent with the expression of ATPase6,ATPase8 mRNA after 12h.Conclusions:（1） Our studies showed that there were mtDNA encoding ATPase6,ATPase8 changes of hepatocytes in rats liver transplantation with cold preservation-refusion,suggesting that mitochondrial function disorders are related to changdes of mitochondrial DNA encoding ATPase6,ATPase8.（2）The RT-PCR and Western-blot results showed that the expression of mtDNA encoding ATPase6,ATPase8 genes of hepatocytes decreased significantly,and F₀F₁-ATPase protein expression was decreased remarkably,which lead to the ATP levels decreased.The energy deficiency lead to liver injury druing cold preservation-refusion in rats liver transplantation.The injury of liver is more obvious with the prolongation of the cold preservation period.The results suggested that the abnormal expression of mtDNA ATPase6,ATPase8 genes were important causes of disorder of mitochondrial energy metabolism.（3） mtTFA and NRF-1 increased the expression of ATPase6,ATPase8 mRNA, suggest which that of mtTFA and NRF-1 may be important factors in controling the expression of ATPase6,ATPase8 mRNA transcription.更多还原