热缺血供肝胆管耐受冷保存时限的实验研究

Experimental study on tolerance time limits from warm ischemia to cold preservation of liver grafts

目的探讨有热缺血损伤的供肝及胆管组织耐受冷保存的安全时限,为临床肝移植术后早期肝内外胆管不可逆性坏死和原发性移植肝无功能的防范提供实验依据。方法在小型猪同种异体原位肝移植模型上,观察供肝血流阻断10 min后移植肝及胆管组织在不同冷保存时间条件下的形态功能改变及其可逆性。结果热缺血10 min,冷保存时间少于16 h组,动物均存活1周以上,且无早期胆管坏死所致的动物死亡;一旦冷保存时间超过16 h,术后早期胆管坏死的发生率显著上升(P<0．05),出现因胆管坏死、胆漏所致的动物死亡;随着冷保存时间的进一步延长,将出现原发性移植肝无功能所致的术中、术后早期动物死亡, 存活动物均发生胆管坏死。与冷保存16 h以内组比较,16 h以上组术中移植肝胆管组织病理形态学评分和上皮凋亡细胞数显著增高(P<0．05),而Na1-K1-ATP酶及Ca2+-ATP酶活力显著降低(P<0．05),术后丙氨酸氨基转移酶、天冬氨酸氨基转移酶、γ-谷氨酰转肽酶、碱性磷酸酶恢复较慢。相关分析结果显示,术后胆管坏死率与术中移植肝胆管细胞病理形态学评分及凋亡细胞数呈显著正相关(r值分别为0．931、0．972, P值均<0．01),与Na+-K+-ATP酶和Ca2+-ATP酶活力呈显著负相关(r值均为-0．973,P值均<0．01)。结论有热缺血损伤的供肝及胆管组织,在限定的冷保存时间范围内是可以利用的。若热缺血时间在10 min以内,冷保存时间延长至16 h对移植肝胆管组织仍安全,延长至20 h移植肝仍能发挥功能。

Objective To investigate the tolerance time limits from warm ischemia to cold preservation of liver grafts. Methods Orthotopic liver transplantations （OLTs） were performed on Bama miniature swine. Morphological and functional changes of the liver grafts and biliary tracts after 10 minutes of warm ischemia followed by different durations of cold preservation and its reversibility were investigated. Results When the grafts were subjected to 10 minutes of warm ischemia followed by less than 16 hours of cold preservation, all animals could survive 1 week and there was no animal death from biliary necrosis. However, when the cold preservation time exceeded 16 hours, the incidence of biliary necrosis was significantly increased （P 〈 0.05）, and recipient death from bile leaks occurred. With further prolongation of the cold preservation time, primary graft nonfunction and intraoperative or early postoperative deaths occurred and the living animals all developed biliary necrosis. When compared with the less than 16 hours cold preservation group, the morphological scores and apoptosis index of the epithelial cells of bile ducts in grafts after reperfusion were significantly elevated in the more than 16 hours cold preservation group （P 〈 0.05） and the activity of Na^＋-K^＋-ATPase and Ca^2＋- ATPase of bile ducts in grafts were also significantly reduced （P 〈 0.05）. Liver function tests showed that the recoveries of AST, AST, GGT and ALP were quicker in the 16 hours cold preservation group then those over 16 hour preservation ones. Correlation analysis revealed that the incidence of biliary necrosis was significantly correlated with the morphological score （r = 0.972） and with the apoptosis index of the epithelial cells of bile ducts in grafts after reperfusion （r = 0.931） and also correlated negatively （P 〈 0.01） with the activity of Na^＋-K^＋-ATPase （r = -0.973） and Ca^2＋-ATPase （r = -0.973）. Conclusious It is concluded that with 10 minutes of warm ischemia, cold preservation of the grafts should not be longer than 16 hours in order to avoid early biliary necrosis, and the corresponding tolerance time limit of the livers to the cold preservation was less than 2 hours.