课题背景：肝移植已成为各种终末期肝脏疾病的有效治疗方法。如何减轻供肝的缺血再灌注损伤，一直是研究的热点。课题建立封闭群大鼠同种异体原位肝移植模型，观察对比银杏叶提取物、左旋精氨酸及不同缺血预处理模式对供肝冷缺血再灌注损伤的保护作用。

应用要点：文章探讨了不同冷保存再灌注时间下移植肝脏肝细胞、肝窦内皮细胞及枯否细胞的损伤情况，发现肝窦内皮细胞的损伤及枯否细胞的激活明显早于并重于肝细胞出现，为在冷保存再灌注损失防护过程中，针对肝窦内皮细胞及枯否细胞进行早期干预，从而减轻移植肝脏的冷保存再灌注损失提供了理论依据。这已在作者的其他课题中得到证实。

同行评价：肝移植术后出现原发性移植肝无功能和移植肝功能恢复延迟严重影响肝移植远期效果，其中冷保存及再灌注损伤是常见的原因之一。实验观察大鼠移植肝脏冷保存及再灌注损伤对肝细胞、肝窦内皮细胞及枯否细胞的损伤，并得出后两者的受损时间早于前者的结论，对于提高供体质量有着深远的意义

摘要
目的: 冷保存及再灌注损伤是决定移植物功能及受者存活率的重要因素之一，这与供肝冷保存再灌注过程中枯否细胞的活化及肝窦内皮细胞的损伤密切相关。建立大鼠同种异体原位肝脏移植模型，比较不同冷保存再灌注时间下供肝肝细胞、肝窦内皮细胞及枯否细胞的损伤情况。
方法：①实验于2005-12/2006-12在河南省实验动物中心及郑州大学第一附属医院肝移植实验室完成，实验方法符合动物伦理学要求。②选用Wistar大鼠63只，采用随机数字表法取7只大鼠为正常对照组，另外56只平分为供体组与受体组，两两配对后再分为供肝冷保存3 h，6 h，8 h和12 h组（每组供、受体各7只）。各冷保存组用4 ℃ Euro-collins液灌注和保存供肝，之后行同种异体原位肝移植。③于受体门脉复流后15，30，60 min检测各组大鼠肝窦内皮细胞损伤指标血清透明质酸、枯否细胞激活指标肿瘤坏死因子α含量及肝细胞损伤指标丙氨酸氨基转移酶、天冬氨酸氨基转移酶活性；观察受体门脉复流后60 min移植肝肝细胞、肝窦内皮细胞及枯否细胞的超微结构变化。
结果：63只大鼠均顺利完成模型建立，术中无异常死亡。①各冷保存组在门脉复流后15，30，60 min血清透明质酸、肿瘤坏死因子α含量均高于正常对照组（P < 0.05），并且随冷保存时间延长依次升高。②各冷保存组在门脉复流后15 min血清丙氨酸氨基转移酶、天冬氨酸氨基转移酶活性与正常对照组无显著性意义（P > 0.05）；在门脉复流30 min后血清丙氨酸氨基转移酶、天冬氨酸氨基转移酶活性高于正常对照组（P < 0.05）。③冷保存3 h及6 h组肝细胞结构基本正常，冷保存8及12 h组出现结构变化；而各冷保存组中，肝窦内皮细胞均有损伤并较肝细胞为重，枯否细胞均有激活表现。
结论：供肝冷保存再灌注过程中，肝窦内皮细胞的损伤及枯否细胞的激活明显早于并重于肝细胞损伤。
关键词：肝移植；组织供者；器官保存；再灌注损伤；肝细胞；枯否氏细胞

宋燕，孙振涛，张弓，李捷，郑守华，张水军.供肝冷保存再灌注过程中肝细胞、肝窦内皮细胞及枯否细胞损伤的比较[J].中国组织工程研究与临床康复，2008，12(5):823-826 [www.zglckf.com/zglckf/ejournal/upfiles/08-5/5k-823(ps).pdf]

Injury to hepatocytes, sinusoidal endothelial cells and Kupffer cells caused by cold preservation and reperfusion in rat liver donors

Abstract

AIM：Cold preservation and reperfusion injury are crucial for graft function and recipient survival, which is closely related to the injury of sinusodial endothelial cells (SEC) and activation of Kupffer cells during different hepatic cold preservation and reperfusion period in rat liver donors. This study established rat models of homologous orthotopic liver transplantation and compare the injuries of hepatocyte, SEC and KC during different hepatic cold preservation and reperfusion period in rat liver donors.
METHODS: ①Experiments were performed at the Henan Experiment Animal Center and Liver Transplantation Laboratory of First Affiliated Hospital of Zhenzhou University from December 2005 to December 2006. Experimental procedures were accorded with the Animal Ethical Standards. ②Sixty-three Wistar rats were randomly allocated into cold preservation 3-hour group, cold preservation 6-hour group, cold preservation 8-hour group, cold preservation 12-hour group, 14 rats in each group, 7 as donors and 7 as recipients, and the 7 else was the normal control group. Liver grafts in cold preservation groups were flushed with and preserved in 4 ℃ Euro-collins solution, the cold preservation time was 3, 6, 8, and 12 hours, respectively. Then orthotopic liver transplantation was performed. ③At 15, 30 and 60 minutes after portal vein reperfusion, blood samples were obtained to determine alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA) and tumor necrosis factor (TNF)-α. At 60 minutes after portal vein reperfusion, the ultrastructure of hepatocyte, liver SEC and Kupffer cells were observed.
RESULTS: All of the sixty-three rats were included in the final results, no death. ①At 15, 30 and 60 minutes after portal vein reperfusion, the levels of HA and TNF-α in cold preservation groups were significantly higher than in normal control group (P < 0.05); and the levels of HA and TNF-α were all increased gradually along with the prolongation of cold preservation. ②At 15 minutes after portal vein reperfusion, the level of ALT and AST in all cold preservation groups had no difference compare with those in normal control group (P > 0.05). But at 30 minutes, the levels of ALT and AST in all cold preservation groups were significantly higher than in normal control group (P < 0.05). ③There was almost no hepatocyte injury except in cold preservation 8-hour and 12-hour groups, but SEC injury appeared and Kupffer cells activated in all cold preservation groups, and the injury of SEC were more severe than in hepatocytes.
CONCLUSION: During cold preservation and reperfusion, the appearance of SEC injury and Kupffer cell activation is earlier and more severe than hepatocytes.

Song Y, Sun ZT, Zhang G, Li J, Zheng SH, Zhang SJ.Injury to hepatocytes, sinusoidal endothelial cells and Kupffer cells caused by cold preservation and reperfusion in rat liver donors.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(5):823-826(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-5/5k-823(ps).pdf]