Infectious and immunological evaluation of xenogeneic extracorporeal liver perfusion and preclinical study

本研究的目的是在临床应用体外肝脏融合作为肝辅助方法之前，检测和分析猪肝与非人灵长类动物之间直接交叉循环的危险因素。1)建立异种体外肝脏长期灌注的最佳条件。与该系统直接交叉循环可延长急性肝衰竭模型动物的存活时间。2)健康狒狒直接连接到ECLP系统。交叉循环持续10小时，发生严重的肉眼溶血，2天后死于急性肾衰竭。宏观上的溶血应该归因于体液因子介导的猪肝移植内皮细胞损伤导致的红细胞直接破碎。然而，少于6小时的交叉循环没有引起溶血，狒狒存活了一年多，没有任何器官功能障碍。3)当狒狒与使用人类腐烂加速因子（hDAF）转基因猪肝的ECLP直接交叉循环时，很少发生溶血，允许交叉循环21±5.8小时（n=5）。实验结束时，所有狒狒都还活着，没有出现任何严重的并发症。在灌注期间，所有猪肝的胆汁生成都得到了保存。4)为了评估猪内源性逆转录病毒（PERV）感染的风险，我们在接触猪器官6个月后，用hDAF猪肝对狒狒进行ECLP治疗后的临床和遗传学观察。在ECLP早期，狒狒外周血中检测到PERV序列和猪特异性着丝粒序列，但在1周内均消失。ECLP后1个月、3个月和6个月未检测到PERV或着丝粒序列。未出现猪细胞微嵌合现象，PERV未发生感染。这些结果表明，使用hDAF转基因猪肝的ECLP具有良好的性能，并且有望用于肝辅助装置。少

The aim of this study was to detect and analyze risk factors of direct cross-circulation between porcine liver and non-human primates prior to a clinical application of extracorporeal liver per fusion as a liver assist method.1) We established the optimized condition for ling-term xenogeneic extracorporeal liver perfusion. The survival of model animals induced acute liver failure prolonged by direct cross-circulation with this system.2) Healthy baboons were directly connected to the ECLP system. When cross-circulation was continued for 10 hours, severe macroscopic hemolysis occurred and the baboon died of acute renal failure two days later. The macroscopic hemolysis should be attributed to direct erythrocyte fragmentation resulting from humoral factor-mediated endothelial cell injury in the porcine liver graft. However, cross-circulation for less than 6 hours did not induced hemolysis, and baboons survived for more than one year without any organ dysfunction.3) When direct cross-circul … More ation between baboons and ECLP using human decay accelerating factor (hDAF) transgenic pig livers, little hemolysis occurred, allowing a cross-circulation for 21 +/- 5.8 hours (n=5). At the end of these experiments, all baboons were alive and did not show any serious complications. Through the perfusion period, bile production of the pig livers wag preserved in all cases.4) In order to evaluate the risk of porcine endogenous retrovirus (PERV) infection, we observed baboons clinically and genetically after ECLP treatment using hDAF pig livers for 6 months after an exposure to the pig organ. The PERV sequences and the pig-specific centromeric sequences were detected in the peripheral blood of baboons during the early time of ECLP, but both disappeared within 1 week. No PERV or centromeric sequences were detected at 1 month, 3 months and 6 months after ECLP. There was no emergence of pig-cell microchimerism, and PERV did not infect.These results indicate that an ECLP using an hDAF transgenic pig liver gives good performance, and shows promise for use in a liver assist device. Less