Natori S, Selzner M, Valentino K L, Fritz L C, Srinivasan A, Clavien P A, Gores G J
Division of Gastroenterology and Hepatology, Mayo Clinic Rochester, MN 55905, USA.
Transplantation. 1999 Jul 15;68(1):89-96. doi: 10.1097/00007890-199907150-00018.
Cold ischemia/warm reperfusion (CI/WR) liver injury remains a problem in liver transplants. Sinusoidal endothelial cells (SEC) are a target of CI/WR injury, during which they undergo apoptosis. Because caspase proteases have been implicated in apoptosis, our aim was to determine whether liver CI/WR injury induces a caspase-dependent apoptosis of SEC.
Rat livers were stored in the University of Wisconsin (UW) solution for 24 hr at 4 degrees C and reperfused for 1 hr at 37 degrees C in vitro. Apoptosis was quantitated using the TUNEL assay, and caspase 3 activation determined by immunohistochemical analysis. Rat liver orthotopic liver transplants (OLT) were also performed using livers stored for 30 hr.
Terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) positive hepatocytes were rare and did not increase during CI/WR injury. In contrast, TUNEL positive SEC increased 6-fold after reperfusion of livers stored under cold ischemic conditions, compared with controls or livers stored but not reperfused. Immunohistochemical analysis demonstrated active caspase 3 only in endothelial cells after CI/WR injury. When IDN-1965, a caspase inhibitor, was given i.v. to the donor animal and added to UW solution and the reperfusion media, TUNEL positive endothelial cells were reduced 63+/-11% (P<0.05). Similarly, the duration of survival after OLT was significantly increased in the presence of the inhibitor.
During liver CI/WR injury: 1) selective apoptosis of endothelial cells occurs; 2) caspase 3 is activated only in endothelial cells; and 3) a caspase inhibitor reduces endothelial cell apoptosis and prolongs animal survival after OLT. The pharmacologic use of caspase inhibitors could prove useful in clinical transplantation.
冷缺血/温再灌注（CI/WR）肝损伤仍是肝移植中的一个问题。肝窦内皮细胞（SEC）是CI/WR损伤的靶点，在此过程中它们会发生凋亡。由于半胱天冬酶蛋白酶与凋亡有关，我们的目的是确定肝CI/WR损伤是否会诱导SEC发生半胱天冬酶依赖性凋亡。
将大鼠肝脏在4℃下于威斯康星大学（UW）溶液中保存24小时，然后在37℃下体外再灌注1小时。使用TUNEL法对凋亡进行定量，并通过免疫组织化学分析确定半胱天冬酶3的激活情况。还使用保存30小时的肝脏进行大鼠原位肝移植（OLT）。
末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）阳性肝细胞很少见，并且在CI/WR损伤期间没有增加。相比之下，与对照组或仅保存但未再灌注的肝脏相比，在冷缺血条件下保存的肝脏再灌注后半胱天冬酶3阳性SEC增加了6倍。免疫组织化学分析显示，CI/WR损伤后仅在内皮细胞中有活性半胱天冬酶3。当将半胱天冬酶抑制剂IDN-1965静脉注射给供体动物，并添加到UW溶液和再灌注培养基中时，TUNEL阳性内皮细胞减少了63±11%（P<0.05）。同样，在存在抑制剂的情况下，OLT后的存活时间显著延长。
在肝CI/WR损伤期间：1）发生内皮细胞的选择性凋亡；2）半胱天冬酶3仅在内皮细胞中被激活；3）半胱天冬酶抑制剂可减少内皮细胞凋亡并延长OLT后动物的存活时间。半胱天冬酶抑制剂的药物应用在临床移植中可能会被证明是有用的。
