Development of the supercooling preservation method for transplant liver graft and analysis of the mechanism of cold preservation injury

过冷很可能使移植器官具有更好的保存性。为了改进肝组织的保存方法，我们尝试了低温非冷冻(SZNF)保存方法。为了将SZNF保存应用于临床，我们使用从细菌^1中分离的抗核蛋白(ANP)作为抗冻剂(AFA)来预防脑震荡器官冻结。最近的研究表明，肝窦内皮细胞(SEC)损伤是冷保存/再灌注损伤的主要靶点，SEC的凋亡起着至关重要的作用。为了保护SECS免受过冷保存损伤，我们使用化学上稳定的抗坏血酸衍生物抗坏血酸2-葡萄糖苷(AA-2G)作为抗氧化剂。在本研究中，我们研究了SECS在超低温缺血损伤中的作用以及在威斯康星大学(UW)溶液中使用ANP和AA-2G联合保存SZNF的效果。SZNF肝脏保存需要解决的问题是过冷增强了更强的低温特异性损伤。在本研究中，由于SECS损伤的微循环障碍，SZNF本身并不能达到较好的移植肝保存效果，活性氧和自由基在冷缺血/再灌流损伤时SECS的凋亡中起着重要作用。AA-2G作为自由基清除剂可减轻过冷增强的SECs的肝窦损伤和抑制细胞凋亡。AA-2G对SECs损伤的保护作用是移植肝过冷保存的结果。SZNF联合ANP和AA-2G的肝脏保存效果优于传统的冷保存

Supercooling is likely to achieve superior preserability of graft organs. In order to improve the preservation procedure of liver grafls, we attempted the subzero nonfreezing (SZNF) storage methods. In consideration of applying the SZNF storage to clinical use, we employed anti-nucleating protein (ANP), isolated from bacterium^1, as anti-freezing agent (AFA) for the prevention of concussive organ freezing.Recent studies showed that the sinusoidal endothelial cell (SEC) damages are the primary targets in cold preservation/ reperfusion injury and the apoptosis of SECs plays a critical role. For protection of SECs against supercooling preservation injury, we used ascorbic acid 2-glucoside (AA-2G) as antioxidant which is chemically stable ascorbic acid derivative.In this research, we investigated the participation of SECs apoptosis in supercold iscbemic injuiy and the efficacy of the SZNF liver storage using ANP with AA-2G in the University of Wisconsin (UW) solution.In our previous study the SZNF storage of isolated SECs failed to show hs advantages as compared with hepalocytes(HC). The problem to be solved for the SZNF liver storage is the stronger hypothermia-specific damage enhanced by supercooling. In this study the SZNF condition by itself could not achieve the better preservavility of HCs in transplanted liver due to the microcirculatoiy disturbance by SECs damage.The reactive oxygen species and free radicals appeared to play an important role in apoptosis of SECs upon cold ischemia / reperfbsion injury. The usage of AA-2G as a free radical scavenger reduced the sinusoidal damages and inhibited apoptosis in SECs enhanced by supercooling. The protective effect of AA-2G against SECs damage led to the better preservavilily of HCs as a consequence of supercooling storage in transplanted liver.The SZNF liver storage with ANP and AA-2G had a potency to surpass the conventional cold preservation