Cao L, Li Y, Cheng F, Li S, Long D
Lab of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Guoxuexiang 37, Chengdu 610041, People's Republic of China.
Transplant Proc. 2006 Sep;38(7):2207-9. doi: 10.1016/j.transproceed.2006.06.014.
Ischemia-reperfusion injury (IRI) is a key factor that contributes to early and late dysfunction of liver graft. Although we have known that hepatocytes express death receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), the effects of TRAIL on hypoxia/reoxygenation (H/R)-mediated apoptosis are unclear. This study sought to examine the effects of H/R on TRAIL cytotoxicity, as a cause of primary hepatic graft dysfunction, delayed graft refunction, and chronic graft dysfunction.
Using an hepatocyte H/R model in vitro to mimic IRI in the grafted liver, normal human hepatocytes HL-7702 were exposed to hypoxia for 5 hours then reoxygenated for 0, 2, 4, 6, or 20 hours. In another experiment, hepatocytes were exposed to hypoxia for 0, 2, 4, 8, or 20 hours. Expressions of TRAIL-R2/Death receptor 5 (DR5) mRNA were measured by semiquantitative reverse-transcriptase polymerase chain reactions. After 16 hours of hypoxia, human hepatocytes were treated with TRAIL in different concentrations for 5 hours. The death of hepatocytes was confirmed by flow cytometer and methyl thiazolyl tetrazolium analysis.
After 5-hour hypoxia, the expressions of DR5 mRNA increased at all times of reoxygenation. DR5 mRNA was up-regulated from 0 hour after reoxygenation, reaching a peak value at 2 hours after reoxygenation compared with the normoxia cultured cells. Moreover, DR5 mRNA was up-regulated gradually following prolonged hypoxia. TRAIL-mediated cell killing was concentration-dependent being greater in the hypoxia treatment group compared to the normoxia group.
H/R up-regulated the expression of DR5 and enhanced TRAIL-mediated apoptosis in an human hepatocyte line. The TRAIL pathway might play a critical role in hepatocyte apoptosis induced by IRI.
缺血再灌注损伤（IRI）是导致肝移植早期和晚期功能障碍的关键因素。尽管我们已经知道肝细胞表达肿瘤坏死因子相关凋亡诱导配体（TRAIL）的死亡受体，但TRAIL对缺氧/复氧（H/R）介导的细胞凋亡的影响尚不清楚。本研究旨在探讨H/R作为原发性肝移植功能障碍、移植肝功能延迟恢复和慢性移植肝功能障碍的原因对TRAIL细胞毒性的影响。
利用体外肝细胞H/R模型模拟移植肝中的IRI，将正常人肝细胞HL-7702暴露于缺氧环境5小时，然后再复氧0、2、4、6或20小时。在另一项实验中，肝细胞暴露于缺氧环境0、2、4、8或20小时。通过半定量逆转录聚合酶链反应检测TRAIL-R2/死亡受体5（DR5）mRNA的表达。缺氧16小时后，用人肝细胞分别用不同浓度的TRAIL处理5小时。通过流式细胞仪和甲基噻唑基四氮唑分析确认肝细胞死亡。
缺氧处理5小时后，复氧各时间点DR5 mRNA表达均增加。与常氧培养细胞相比，复氧后0小时DR5 mRNA上调，复氧后2小时达到峰值。此外，随着缺氧时间延长，DR5 mRNA逐渐上调。TRAIL介导的细胞杀伤具有浓度依赖性，缺氧处理组比常氧组更明显。
H/R上调人肝细胞系中DR5的表达并增强TRAIL介导的细胞凋亡。TRAIL通路可能在IRI诱导的肝细胞凋亡中起关键作用。
