常温机械灌注对大鼠心脏死亡器官捐献供肝微循环保护作用的研究

目的 探讨常温机械灌注(normothermic machine perfusion,NMP)对大鼠心脏死亡器官捐献(donation after circulatory death,DCD)供肝微循环发挥的作用.方法 采用夹闭SD大鼠胸主动脉热缺血30 min获取DCD供肝;在体外建立大鼠NMP系统.根据不同的保存供...

Study on the protective effect of normothermic machine perfusion on hepatic microcirculation in rat DCD donors

Objective To explore the effect of normothermic machine perfusion(NMP)on the microcirculation of rat donation after circulatory death(DCD)liver.Methods DCD livers were obtained by clipping SD rat thoracic aorta for 30 minutes as warm ischemia;rat NMP system was established in vitro.According to different methods of preservation of the donor livers,the experiment was divided into:Normal group(n=6),serum and livers were retained for use;NMP group(n=30),livers were collected after 4,6,and 8 h,outflow and inflow perfusate was collected at 0,2,4,6 and 8 h after infusion for testing;static cold storage(SCS)group(n=6),the livers were flushed out of the blood with 20 ml 4℃UW solution and were collected after 6 h of SCS in UW solution at 4℃.Liver function in outflow perfusate was detected by biochemical methods;liver tissue histopathology was observed by hematoxylin-eosin staining;hepatocyte ultrastructure was observed by transmission electron microscopy;hepatocyte apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling;endothelin-1,endothelial nitric oxide synthase(eNOS),inducible nitric oxide synthase(iNOS),von Willebrand factor(vWF),intercellular adhesion molecule(ICAM-1)and intervascular adhesion molecule-1(VCAM-1)expression were detected by Western blot.Results Compared with SCS,NMP significantly improved the histological damage of DCD donor livers,the Suzuki’s score of NMP group(3.40±0.55)is significantly lower than that of SCS group(7.00±0.71,F=229.75,P<0.05);reduced hepatocytes apoptosis,the number of apoptotic cells in the NMP group(9.80±1.48)was significantly lower than that in the SCS group(33.40±4.39,F=166.58,P<0.05);meanwhile,NMP could repair liver mitochondrial damage,the number of irreversible damaged mitochondria in the NMP group(1.60±0.55)was significantly lower in the SCS group(2.80±0.45,F=36.29,P<0.05).It was further found that NMP could improve DCD liver through:①Inhibiting intercellular adhesion and improving endothelial cell damage;compared with SCS,NMP significantly inhibited the expression of ICAM-1(F=1728.45,P<0.05),VCAM-1(F=254.72,P<0.05)and vWF(F=595.30,P<0.05)in the liver.②Improving liver ET-1/NOS balance and microcirculation perfusion;compared with SCS,NMP significantly inhibited the expression of ET-1(F=1372.51,P<0.05)and iNOS(F=1102.20,P<0.05)in the liver and promoted the expression of eNOS(F=271.66,P<0.05).Conclusion NMP could improve the microcirculation of DCD donor liver and improve the quality of donor liver.The mechanism may rely on its role in inhibiting intercellular adhesion,improving sinusoidal endothelial injury and microcirculation perfusion.