移植肝冷保存-再灌注中肝细胞线粒体DNA ATPase8基因表达改变与细胞凋亡的研究

Effects of the change in the expression of mt DNA ATP8 on hepatocytes apoptosis in rat liver transplantation with cold preservation-refusion

目的探讨移植肝冷保存-再灌注中肝细胞线粒体DNA ATPase8基因表达改变与细胞凋亡的关系。方法 (1)Wistar大鼠186只,采用改良"二袖套法"复制大鼠肝移植模型,动物随机分为A组(冷保存30min组),B组(冷保存6h组),C组(冷保存12h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)观察线粒体形态变化,检测各组大鼠肝脏SOD、ATP含量和肝细胞凋亡数量。(3)采用RT-PCR检测mtDNA ATPase8 mRNA表达。结果冷保存-再灌注后早期(12h),A、B、C3组SOD、ATP含量、ATPase8 mRNA表达均降低,C组显著低于A组、B组(P<0.05),并且C组肝细胞线粒体受损较重,凋亡数量明显多于A组、B组(P<0.05)。再灌注24h后A、B、C3组SOD、ATP含量、ATPase8 mRNA表达升高,线粒体损伤减轻,肝细胞凋亡减少。结论冷保存-再灌注后ATPase8基因表达量和ATP含量减少,细胞凋亡增多,并且冷保存时间的延长,再灌注后变化越显著,提示ATPase8基因表达异常可能是影响肝细胞凋亡的主要原因之一。

Objective study effects of the change in the expression of mt DNA ATP8 on hepatocytes apoptosis in rat liver transplantation with cold preservation-refusion. 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：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 3rd,5th,7th days after operation. The ATP,SOD and apoptosis of liver were observed in each group,mtDNA encoding ATPase8 mRNA were determined by RT-PCR. Results The content of ATP ,SOD and the expression of ATPase8 mRNA were decreased in groups A,B and C on 12 h after operation,group C were the lowest in when compared with groups A and B （P0.05）. The amount ofhepatocytes apoptosis in group C were more than that of groups A and B （P0.05）. The expression of ATPase8 mRNA,ATP and SOD increased and the amount of hepatocytes apoptosis were decreased after 24 h in groups A,B and C. Conclusion mtDNA encoding ATPase8 genes of hepatocytes decreased significantly,which lead to the ATP levels decreased and the amount of hepatocytes apoptosis increased. The injury of liver is more obvious with the prolongation of the cold preservation period. The results suggested that the abnormal expression of mtDNA ATPase8 genes were important causes of hepatocytes apoptosis.