新型携氧纳米粒人工灌注液用于猪心死亡后供肝常温灌注保存

Application of a novel artificial perfusate based on oxygencarrying nanoparticles in normothermic machine perfusion for porcine liver preservation after cardiac death

目的:探讨以全氟萘烷白蛋白纳米粒作为携氧物的人工灌注液用于猪心死亡后供肝常温机器灌注(NMP)保存的效果。方法:制备全氟萘烷白蛋白纳米粒,按照5%白蛋白(w/v)配成人工灌注液并测试其载氧能力。16只长白猪的肝脏在热缺血1h后取出,随后均分为四组,用不同的保存方法连续保存24h:UW液冷保存(静态冷保存组)及动物自体血(全血NMP组)、含5%白蛋白(w/v)的平衡盐液(非携氧纳米粒NMP组)和全氟萘烷白蛋白纳米粒灌注液(携氧纳米粒NMP组)保存。灌注开始后每4h监测血流动力学、代谢、灌注液和胆汁的生化指标。在保存前、保存12和24h后分别收集肝组织标本行苏木精-伊红染色和TUNEL检测观察。结果:每100mL人工灌注液中全氟萘烷白蛋白纳米粒的载氧能力为6.94μL/mmHg(1mmHg=0.133kPa)。灌注过程中携氧纳米粒NMP组和全血NMP组肝动脉和门静脉循环阻力保持稳定,且携氧纳米粒NMP组循环阻力低于全血NMP组;两组灌注液乳酸浓度均在8h内降至正常范围;灌注开始后各监测时间点携氧纳米粒NMP组和全血NMP组灌注液丙氨酸转氨酶、天冬氨酸转氨酶水平和累计胆汁产量差异均无统计学意义(均P>0.05)。灌注24h后全血NMP组和携氧纳米粒NMP组的肝组织Suzuki评分均低于静态冷保存组和非携氧纳米粒NMP组(均P<0.05),灌注12、24h后全血NMP组和非携氧纳米粒NMP组TUNEL染色阳性细胞数明显高于携氧纳米粒NMP组和静态冷保存组(均P<0.05)。结论:含全氟萘烷白蛋白纳米粒的人工灌注液可以满足NMP保存时猪心死亡后供肝的氧耗需求,其保存效果与血液相当,在改善组织微循环和减轻缺血再灌注损伤方面可能更具优势。

Objective:To investigate the efficacy of a novel artificial perfusate based on oxygen-carrying perfluoronaphthalene-albumin nanoparticles in normothermic machine perfusion(NMP)for preservation of porcine liver donation after cardiac death.Methods:Artificial perfusate with perfluoronaphthalene-albumin nanoparticles was prepared at 5%albumin(w/v)and its oxygen carrying capacity was calculated.The livers of 16 Landrace pigs were isolated after 1 h of warm ischemia,and then they were divided into 4 groups and preserved continuously for 24 h with different preservation methods:cold preservation with UW solution(SCS group),NMP preservation by whole blood(blood NMP group),NMP preservation by artificial perfusate without nanoparticles(non-nanoparticles NMP group)and NMP preservation by artificial perfusate containing nanoparticles(nanoparticles NMP group).Hemodynamics,tissue metabolism,biochemical indices of perfusate and bile were monitored every 4 h after the beginning of NMP.Liver tissue samples were collected for histological examination(HE and TUNEL staining)before preservation,12 h and 24 h after preservation.Results:The oxygen carrying capacity of nanoparticles in 100 mL artificial perfusate was 6.94μL/mmHg(1 mmHg=0.133 kPa).The hepatic artery and portal vein resistance of nanoparticles NMP group and blood NMP group remained stable during perfusion,and the vascular resistance of nanoparticles NMP group was lower than that of blood NMP group.The concentration of lactic acid in the perfusate decreased to the normal range within 8 h in both nanoparticles NMP group and blood NMP group.There were no significant differences in accumulated bile production,alanine aminotransferase and aspartate aminotransferase in perfusate between nanoparticles NMP group and blood NMP group(all P>0.05).After 24 h perfusion,the histological Suzuki score in blood NMP group and nanoparticles NMP group was lower than that in SCS group and non-nanoparticles NMP group(all P<0.05),and the quantities of TUNEL staining positive cells in blood NMP group and non-nanoparticles NMP group was higher than those in nanoparticles NMP group and SCS group 12 h and 24 h after preservation(all P<0.05).Conclusion:Artificial perfusate based on oxygen-carrying nanoparticles can meet the oxygen supply requirements of porcine livers donation after cardiac death during NMP preservation,and it may has superiorities in improving tissue microcirculation and alleviating ischemia-reperfusion injury.