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  

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

作　　者：杨柳[1] 曹欢 孙东 侯宾 林玲[1] 宋红丽[2] Yang Liu;Cao Huan;Sun Dong;Hou Bin;Lin Ling;Song Hongli(The First Central Clinical College,Tianjin Medical University,Tianjin 300192,China;Department of Organ Transplantaion,Tianjin First Central Hospital,Tianjin Key Laboratory of Organ Transplantation,Key Laboratory of Transplant Medicine,Chinese Academy of Medical Sciences,Key Laboratory of Critical Care and Emergency Medicine of the National Health Commission,Tianjin 300192,China.)

机构地区：[1]天津医科大学一中心临床学院,天津300192 [2]天津市第一中心医院器官移植科,天津市器官移植重点实验室,中国医学科学院移植医学重点实验室,国家卫生健康委员会危重病急救医学重点实验室,天津300192

出　　处：《实用器官移植电子杂志》2021年第2期120-125,共6页Practical Journal of Organ Transplantation(Electronic Version)

基　　金：国家临床重点专科建设项目经费资助国家重点项目(2012--649)。

摘　　要：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。根据不同的保存供肝方式,将实验分为:正常(Normal)组(n=6),留取血清及肝脏待用;NMP组(n=30),保存4、6和8 h后收集肝脏标本,灌注后0、2、4、6 h和8 h收集流入道、流出道灌注液待检;静态冷保存(SCS)组(n=6),肝脏以20 ml 4℃UW液冲出肝内血液,并于UW液中4℃SCS 6 h后收集肝脏标本。采用生物化学方法检测流出道灌注液的肝功能;HE染色观察肝组织病理学改变;透射电镜观察肝细胞超微结构;TUNEL检测肝脏细胞凋亡情况;Western blot检测肝脏组织内皮素-1(endothelin-1,ET-1)、内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、血管性血友病因子(von Willebrand factor,vWF)、细胞间黏附分子(intercellular adhesion molecule-1,ICAM-1)和血管间黏附分子-1(intervascular adhesion molecule-1,VCAM-1)的表达情况。结果与SCS比较,NMP能显著改善DCD供肝组织学损伤,Suzuki评分NMP组(3.40±0.55)显著低于SCS组(7.00±0.71,F=229.75,P<0.05);减轻肝细胞凋亡,NMP组凋亡细胞数(9.80±1.48)显著低于SCS组(33.40±4.39,F=166.58,P<0.05);同时能修复肝细胞线粒体损伤,NMP组不可逆损伤线粒体数量(1.60±0.55)显著低于SCS组(2.80±0.45,F=36.29,P<0.05)。进一步发现,NMP可改善DCD肝脏微循环:①抑制细胞间黏附,改善内皮细胞损伤,与SCS比较,NMP显著抑制肝内ICAM-1(F=1728.45,P<0.05)、VCAM-1(F=254.72,P<0.05)和vWF(F=595.30,P<0.05)的表达;②改善肝脏ET-1/NOS平衡和微循环灌注,与SCS相比,NMP显著抑制肝内ET-1(F=1372.51,P<0.05)、iNOS(F=1102.20,P<0.05)的表达,促进eNOS(F=271.66,P<0.05)的表达。结论NMP能够改善大鼠DCD供肝质量,其机制可能通过抑制细胞间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 si

关 键 词：心脏死亡器官捐献 常温机械灌注 肝脏微循环 内皮素-1 一氧化氮合酶 

分 类 号：R654.2[医药卫生—外科学]