Abstract:

obtained by clippingSD rat thoracic aorta for 30 minutes as warm ischemia；rat NMP system was established in vitro. According to differentmethods 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 perfusatewas 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 ；hepatocyteapoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling ；endothelin-1，endothelialnitric 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 byWestern blot. Results Compared with SCS，NMP significantly improved the histological damage of DCD donorlivers，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 NMPcould improve DCD liver through: ① Inhibiting 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. ② Improving 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 endothelialinjury and microcirculation perfusion. 

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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 ；Westernblot 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黏附，改善窦内皮损伤及微循环灌注发挥保护作用的。

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