大鼠DCD供肝热缺血时间对移植肝的影响
Effect of warm ischemia time of donor liver from donation after cardiac death on transplant liver in rats
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摘要:目的 评价大鼠心脏死亡器官捐献(DCD)供肝热缺血时间对移植肝的影响。方法 建立大鼠DCD供肝原位肝移植模型。根据供肝获取前经历心脏停搏时间的不同, 将54只受体大鼠分为3组:对照组(W0组, 未经历热缺血)、热缺血10 min组(W10组)、热缺血20 min组(W20组), 每组18只。各组大鼠分别于术后1、3、7 d检测血清肝功能丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST), 电子显微镜观察肝细胞及其细胞器, 用蛋白质免疫印迹(Western blot)法检测肝组织细胞色素C(Cyt C)和凋亡诱导因子(AIF)的蛋白表达水平。结果 随着热缺血时间的延长, 肝移植术后1 d, 各组大鼠ALT和AST水平急剧上升, 与W0组相比, W10组和W20组的ALT和AST水平均明显升高(均为P < 0.05)。术后3 d, 各组大鼠ALT和AST水平均稍稍下降, 与W0组相比, W10组和W20组的ALT和AST水平仍明显较高(均为P < 0.05)。术后7 d, 各组大鼠ALT和AST水平均再次上升, 与W0组相比, W10组和W20组的ALT和AST水平升高更显著(均为P < 0.05)。电子显微镜观察发现, 随着热缺血时间的延长, 肝细胞水肿程度逐渐加重, 内质网扩张逐渐加重, 线粒体损伤程度加重, W20组尤为严重。术后1 d, 3组大鼠肝组织中的AIF和Cyt C蛋白表达水平的比较, 差异均无统计学意义(均为P > 0.05)。术后3、7 d, 与W0组比较, W10组和W20组的AIF与Cyt C蛋白表达水平均较高, 差异均有统计学意义(均为P < 0.05), 且W20组的AIF与Cyt C蛋白表达水平均高于W10组(均为P < 0.05)。结论 DCD供肝热缺血主要损伤肝细胞, 随着热缺血时间的延长, 移植肝肝细胞内线粒体和其他细胞器形态、及线粒体凋亡相关蛋白水平均出现明显变化。Abstract:Objective To evaluate the effect of warm ischemia time of donor liver from donation after cardiac death (DCD) on transplant liver in rats.Methods The rat models of orthotopic liver transplantation of DCD donor liver were established.According to the time of cardiac arrest before obtaining the donor livers, 54 recipient rats were evenly divided into three groups:control group (W0 group, no warm ischemia, n=18), 10 min warm ischemia group (W10 group, n=18) and 20 min warm ischemia group (W20 group, n=18).At 1, 3, and 7 d after operation, serum liver function alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were quantitatively measured in rats of each group, liver cells and its organelles were observed by electron microscopy and the expression levels of cytochrome C (Cyt C) and apoptosis inducing factor (AIF) proteins in liver tissues were detected by Western blot in rats of each group.Results The serum levels of ALT and AST in each group were significantly increased at 1 d after liver transplantation along with the prolongation of warm ischemia time.Compared with the W0 group, the serum levels of ALT and AST in the W10 and W20 groups were increased significantly (all P < 0.05).At postoperative 3 d, the serum levels of ALT and AST in each group were slightly decreased.Compared with the W0 group, the serum levels of ALT and AST in the W10 and W20 groups were significantly higher (all P < 0.05).At 7 d after operation, the serum levels of ALT and AST in each group were elevated again.Compared with the W0 group, the serum levels of ALT and AST in the W10 and W20 groups were remarkably increased (all P < 0.05).Electron microscope showed that along with the prolongation of warm ischemia time, the degree of hepatocyte edema was gradually aggravated, the endoplasmic reticular expansion was steadily increased and the degree of mitochondrial damage was aggravated, especially in the W20 group.At postoperative 1 d, the expression level of AIF and Cyt C proteins in the liver tissues did not significantly differ among three groups (all P > 0.05).At 3 and 7 d after operation, compared with the W0 group, the expression levels of AIF and Cyt C proteins were significantly up-regulated in the W10 and W20 groups (all P < 0.05), and the expression levels of AIF and Cyt C proteins in the W20 group were significantly higher than those in the W10 group (both P < 0.05).Conclusions The warm ischemia of the DCD donor liver mainly leads to liver cell injury.Along with the extension of warm ischemia time, significant changes occur in the morphology of mitochondria and other organelles and the expression level of mitochondrial apoptosis-related proteins in transplanted liver cells.
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