线粒体ATP酶6、8基因及编码蛋白表达改变对移植肝脏能量代谢的影响

目的 探讨肝细胞线粒体DNA ATP酶6、ATP酶8基因及编码蛋白F_0F_1-ATP酶表达改变对移植肝能量代谢的影响.方法 采用改良"二袖套法"制作大鼠肝移植模型,随机分为A组(冷保存30 min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12 h、24 h、3 d、5 d、7 d采集标本,检测各组大鼠肝脏ATP含量、mtDNA ATP酶6、ATP酶8mRNA和F0F1-ATP酶蛋白表达.结果 冷保存再灌注早期(12 h),各组ATP酶6、ATP酶8 mRNA、F_0F_1-ATP酶蛋白表达(ng/mg蛋白)和ATP含量(U/L)均降低,C组(0.81±o.08,0.71±0.07,0.47±0.07,0.44 ±0.05)显著低于A组(1.11±0.04,1.07±0.07,0.86±0.15,0.70±0.11)、B组(1.02±0.07,0.90±0. 10,0.65 ±0.07,0.55±0.08)(P<0.05).再灌注24 h后各组ATP酶6、ATP酶8mRNA、F_0F_1-ATP酶蛋白表达和ATP含量增加,c组升高较A组、B组缓慢.结论 ATP酶6、ATP酶8基因表达改变后F_0F_1-ATPase蛋白表达异常.可能是导致能量合成障碍的主要原因.

Altered expression of mitochondrial DNA encoding ATP 6,8 and encoding protein in energy metabolism during cold preservation and reperrusion injury in rat liver transplantation

Objective To study the expression changes of mitoehondrial (mt) DNA encoding ATP6,8 of hepatocytes in rat liver transplantation with cold preservation-reperfusion and explore the relationship between the expression of F_0F_1-ATPase induced by changes of ATP 6,8 genes.Methods Orthotopic liver transplantation was performed in Wistar rats using the cuff technique as described by Kamada with modifications.Rats were randomly divided into four groups:group A (30 min cold preservation),group B(6 h cold preservation),group C(12 h cold preservation)and group D(sham operation).Samples were collected at 12 h,24 h and on Day 3,5 and 7 post-operation.The levels of mtDNA encoding ATPase 6. ATPase 8 mRNA were determined by RT-PCR The expression changes of F_0F_1-ATPase protein were examined by Western blot.The ATP level of liver was observed in each group.Results The expression of ATPase 6,ATPase8 mRNA,F_0F_1-ATPase protein expression(ng/mg protein)and the level of ATP decreased(U/L)in groups A,B and C at 12 h post-operation.The lowest was in group C(0.81±0.08, 0.7l±0.07,0.47 ±0.07,0.44±0.05)when compared with groups A(1.11±0.04,1.07±0.07,0.86±0.15,0.70±0.11)and B(1.02±0.07,0.90±0.10,0.65±0.07,0.55±0.08)(P<0.05).The expression of ATPase 6 ATPase 8 mRNA increased after 24 h in groups A,B and C.The changes of F_0F_1-ATPase protein expression and ATP level were consistent with the expression of ATPase 6.ATPase 8 mRNA.Both ATPase 6 mRNA,ATPase 8 mRNA and F_0F_1-ATPase protein in group C increased slowly compared with groups A and B after 24 h,The mtDNA encoding ATPase 6.ATPase 8 changes of hepatocytes in rats liver transplantation with cold preservation-reperfusion suggested that mitochondrial function disorders are related to the changes of mitochondfial DNA encoding ATPase6.ATPase8.mtDNA encoding ATPase 6, ATPase 8 genes of hepatocytes decreased significantly and F_0F_1-ATPase protein expression decreased remarkably and resulted in the decrease of ATP levels.The energy deficiency led to liver injury during cold preservation-reperfusion in rats liver transplantation.The injury of liver was more obvious with the prolongation of cold preservation period.Conclusion The abnormal expressions of mtDNA ATPase 6.ATPase 8 genes are important causes of the changes of F_0F_1-ATPase protein expression resulting in a disorder of mitochondrial energy metabolism.