自体吞噬参与大鼠肝移植术后原发性肝功能障碍

Participation of Autophagy in Primary Graft Dysfunction after Rat Liver Transplantation

目的应用大鼠原位肝脏移植模型,研究自体吞噬(自噬)如何参与缺血再灌注损伤后肝细胞死亡过程。方法在肝脏UW保存液中4°C保存24h后行大鼠同种原位肝脏移植;渥曼青霉素治疗组(WM-G)为UW保存液中添加渥曼青霉素(100 n M),对照组(UW-G)为UW治疗组:大鼠原位肝移植后观察受体生存率,分别于肝移植后15、30、60及120min检测血液血清谷草转氨酶(AST)及谷丙转氨酶(ALT),肝脏标本行光学显微镜及透射电子显微镜组织形态学分析,免疫印迹方法进行自噬标记分子LC3定量。结果移植15min后,UW-G细胞质中含有大量吞噬小体和吞噬溶酶体的小团肝细胞自肝细胞索分离,分离的肝细胞堵塞肝窦内皮间隙从而引起移植2h后大面积的肝细胞坏死,坏死的肝细胞中包含非活化半胱氨酸天冬氨酸酶-3及非活化半胱氨酸天冬氨酸酶-7的调亡染色阳性核;自体吞噬标志性蛋白LC3-II的表达各组都逐渐增加,移植30min后达到高峰,WM-G可明显提高肝移植受体存活率,明显降低AST及ALT,WM-G移植15min后肝细胞中LC3-II的表达低于UW-G;超微结构显示移植15min后WM-G肝细胞中早期自体吞噬性液泡(AVi)、晚期自体吞噬性液泡(AVd)、自噬溶酶体/溶酶体(包括致密体)的比例明显低于UW-G(P<0.01)。结论自噬参与了肝脏冷缺血再灌注损伤过程,抑制自噬可降低肝移植术后冷缺血再灌注损伤。

Objective To exam the contribution of autophagy in the degeneration procedure of rat hepatocytes after liver transplantation following sustained cold preservation. Methods Rat livers were obtained and preserved in WM( UV solution + Wortmannin) or UW( UW solution) solutions for 24 hours at 4℃,and orthotopic liver transplantation were then undertaken. After liver transplantation,survival rates were calculated.Levels of aspartate aminotransferase( AST) and alanine aminotransferase( ALT) in serum were measured at15,30,60 and 120 min after transplantation. Western blot was used to detect the expression of LC3 and microscopy was used to analyze the morphology of liver tissues. Results Morphological analysis showed that small group of hepatocytes containing large phagosome and phagolysosome started to isolate from hepatic cell cords at the 15 th min after transplant. The isolated hepatocytes leaded to hepatocellular necrosis in 2 hours.Positive staining of Caspase 3 and Caspase 7 were detected,and the expression of LC3- II increased. The expression of LC3- II peaked at 30 min after transplant. Compared with UW- G,WM- G significantly improved survival rates and decreased levels of AST and ALT and LC3- II expression. Importantly,early autophagic vacuoles( AVi),late of autophagy vacuoles( AVd) and the proportion of lysosomal autophagy / lysosomal( including dense body) in liver cells treated with WM- G were significantly lower than those in UW- G( P < 0. 01). Conclusions These data suggested that autophagy involved in Liver ischemia /reperfusion injury and the. inhibition of autophagy can reduce Liver ischemia / reperfusion injury after liver tranplatation.