小鼠肝脏持续低灌注模型的建立及其对热缺血再灌注损伤的耐受性

目的 建立一种稳定的小鼠肝脏持续低灌注模型,并在此基础上研究肝脏持续低灌注对小鼠肝脏热缺血再灌注损伤的影响.方法 选用6～8周龄C57BL/6小鼠建模,将门静脉缩窄至1 mL注射器针头直径,门静脉缩窄后3d、7d、14d和21 d行肝功能及肝脏组织病理学检测;选用稳定的模型小鼠行70％缺血再灌注手术,再灌注3h、24 h、48 h后行肝功能及肝脏组织病理学检测.对照组采用正常C57BL/6小鼠行缺血再灌注手术.结果 小鼠门静脉缩窄术后,丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)均有不同程度的升高,在7d时达到高峰ALT:(60.8±6.2)U/L vs (25.5±2.8) U/L,P＜0.001;AST:(74.9±6.1)U/L vs (39.1±3.2) U/L,P＜0.001),同时H-E染色显示7d时肝细胞损伤最重,并且有较多炎细胞浸润;在21 d时,ALT基本恢复正常水平(P＞0.05),而AST仍高于正常水平(P=0.03).低灌注处理7d的小鼠进行缺血再灌注手术后,肝酶和组织病理学检查显示肝细胞损伤较对照组显著加重,肝酶在再灌注3h达到高峰ALT:(8 217.0±1 111.8) U/L vs(5 597.4±1 015.3) U/L,P=0.004;AST:(8 548.2±1 155.4)U/L vs(5 765.4±956.9)U/L,P=0.003];再灌注48 h时,对照组小鼠ALT和AST均恢复正常,而经过低灌注处理的小鼠肝酶仍高于对照组ALT:(608.8±442.9)U/L vs (47.4±20.1)U/L,P=0.008;AST:(861.8±442.8)U/L vs (70.8±68.3)U/L,P=0.008).结论 成功建立了稳定的小鼠肝脏持续低灌注模型,经持续低灌注处理后的肝脏对热缺血再灌注损伤的耐受能力显著降低,这在一定程度上能够模拟临床上心死亡器官捐献供肝的状况.

Construction of a sustained hepatic hypoperfusion model in mice and its tolerance to hepatic warm ischemia-reperfusion injury

Objective To construct a stable model of sustained hepatic hypoperfusion in mice, and to explore the influence of sustained hepatic hypoperfusion on warm ischemic-reperfusion (IR) injury. Methods C7BL/6 mice aged 6-8 weeks old were chosen for model construction. The portal vein of mice was constricted to the syringe needle with diameter of 1 mL. Liver function and histopathology (H-E staining) were tested on 3, 7, 14 and 21 days after constriction. Hepatic warm IR injury of 70% was performed in stable hepatic hypoperfusion models. Liver function and histopathology were tested again at 3, 24 and 48 hours after reperfusion. Wild type C7BL/6 mice were used as controls. Results After portal vein constriction, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) rose to different extents, and the levels peaked on day 7 (ALT: 60.8±6.2] U/L vs 25.5±2.8] U/L, P<0.001; AST: 74.9±6.1] U/L vs 39.1±3.2] U/L, P<0.001). Meanwhile, H-E staining showed that the most serious damage to hepatocytes and more inflammatory cell infiltration appeared on day 7 after constriction. On day 21, ALT almost returned to the normal level (P>0.05), but AST was still significantly higher than baseline (P=0.03). Compared with the control group, damage of hepatocytes was more severe in mice with 7 day-hypoperfusion precondition after warm hepatic IR. The transaminase levels peaked at 3 h after reperfusion (ALT: 8 217.0±1 111.8] U/L vs 5 597.4±1 015.3] U/L, P=0.004; AST: 8 548.2±1 155.4] U/L vs 5 765.4±956.9] U/L, P=0.003). At 48 h after reperfusion, ALT and AST were back to the normal levels in the control group, but they kept at higher levels in the hypoperfusion precondition group (ALT: 608.8±442.9] U/L vs 47.4±20.1] U/L, P=0.008; AST: 861.8±442.8] U/L vs 70.8±68.3] U/L, P=0.008). Conclusion A stable mouse model of sustained hepatic hypoperfusion has been successfully constructed. The model has decreased tolerance to warm IR injury in hypoperfusion precondition liver, which can partly mimic the liver graft in donation after cardiac death in clinic.