TRAIL/死亡受体5途径在缺氧再给氧诱导人肝细胞凋亡中的作用及相关机制

目的:研究肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis-inducing ligand,TRAIL)/死亡受体5(Death receptor 5,DR5)途径在人肝细胞缺氧/再给氧(Hypoxia/reoxygenation,H/R)损伤中的作用,并探讨其作用机制。方法:在体外建立肝细胞H/R模型,模拟肝脏的缺血再灌注。H/R处理后,培养基中加入不同浓度外源性TRAIL蛋白,噻唑盐比色法(MTT)和流式细胞术检测细胞活力和凋亡率。半定量RT-PCR检测DR5mRNA的表达。结果:以正常人肝细胞为对照,H/R使人肝细胞的DR5 mRNA的表达上调,再给氧2小时达峰值,此后直至再给氧20小时维持在相对高的水平。单纯缺氧6小时不能引起肝细胞的大量凋亡。TRAIL诱导H/R处理后的肝细胞发生凋亡,并呈浓度依赖性。H/R大大增强TRAIL的肝毒性。结论:H/R使DR5在人肝细胞的表达上调,增加TRAIL的肝毒性。TRAIL/DR5途径可能在IRI诱导人肝细胞凋亡过程中发挥重要作用。

TNF- related Apoptosis- inducing Ligand and Death Receptor 5 Mediate the Apoptosis Signaling Induced by Hypoxia/Reoxygenation in Human Hepatocyte Line

Objective:Ischemia and reperfusion activate hepatocyte apoptosis,which is a key factor that contributes to ischemia reperfusion injury(IRI).Although we have known that the expression of death receptors for TNF-related apoptosis-inducing ligand(TRAIL)could be induced on hepatocytes,the effects of hypoxia/reoxygenation(H/R)on TRAIL-mediated apoptosis is unclear.This study aim to investigate the effect of H/R on the expression of death receptor 5(DR5)and TRAIL cytotoxicity,hope to find the causes of hepatic ischemia reperfusion injury.Methods:Using hepatocyte H/R model in vitro to mimic IRI of graft liver,normal human hepatocytes line HL-7702 were exposed to hypoxia condition(1%0_2)for 6 hours,then reoxygenation for 0,2,4,6 and 20 hours separately.Expressions of DR5 mRNA were measured by semi-quantitative RT-PCR.Moreover,human hepatocytes were hypoxia/reoxygenated and treated with TRAIL in different concentration for 5h.,Death of hepatocytes were confirmed by FCM and MTT analysis.Results:Comparing with the normoxia cultured cells,H/R increased the expression of DR5 mRNA in hepatocytes.After 6h hypoxia,the expression of DR5 mRNA increased in all time points of reoxygenation.The expression reached peak value on 2h after reoxygenation and maintained the high level to 20h after reoxygenation.After 6h hypoxia,TRAIL-mediated cell killing was concentration-dependent.Undergoing the TRAIL treatment(50ng/ml-200ng/ml),cell viability reduced and apoptosis rate increased in H/R group comparing with the normoxia group(P<0.01).Conclusion:Hypoxia/reoxygenation up-regulated the expression of DR5 and enhanced TRAIL-mediated apoptosis in human hepatocyte.TRAIL/DR5 pathway might play critical role in hepatocytes apoptosis induced by hypoxia/reoxygenation.