Feng Li, Pang Lili, Guo Yingjia, Ke Nengwen, Li Shengfu, Wei Liang, Li Quansheng, Li Youping
Key Laboratory of Transplant Engineering and Immunology of Health Ministry of China, West China Hospital, Sichuan University, Chengdu, 610041, Sichuan Province, PR China.
Life Sci. 2009 Aug 26;85(9-10):401-7. doi: 10.1016/j.lfs.2009.07.011. Epub 2009 Jul 28.
To investigate whether ischemia/reperfusion (I/R)-induced apoptosis in the bile duct epithelium could be mediated by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors in biliary epithelial cells, we examined the effects of hypoxia/reoxygenation (H/R) on TRAIL cytotoxicity.
Using an H/R model, normal primary human intrahepatic biliary epithelial cells were exposed to hypoxia for 1 h, and then reoxygenated. Expressions of death receptor 4 (DR4) and DR5 mRNA and protein were measured. After 1 h of hypoxia, biliary epithelial cells were treated with TRAIL in different concentrations for 4 h. The death of biliary epithelial cells was confirmed by analysis of apoptosis and methylthiazolyl tetrazolium. The activities of caspase-3 and caspase-8 were determined by fluorometric assay.
Compared with normoxic-cultured cells, the mRNA expressions of DR4 and DR5 were up-regulated from 0 min after reoxygenation, reaching a peak value at 60 min after reoxygenation. The protein expression of DR4 was most intense at 90 min after reoxygenation; the most intense expression of DR5 came at 120 min after reoxygenation. The apoptosis rate increased in the TRAIL treatment group and further increased in the TRAIL plus H/R group, and the effect of concentration-dependent TRAIL-mediated cell killing was more pronounced. Caspase-3 and caspase-8 enzymatic activities after H/R also increased with increased TRAIL concentration.
H/R up-regulated the expression of DR4 and DR5, and enhanced TRAIL-mediated apoptosis in normal human intrahepatic biliary epithelial cells.
为了研究胆管上皮细胞中缺血/再灌注（I/R）诱导的细胞凋亡是否可由肿瘤坏死因子相关凋亡诱导配体（TRAIL）受体介导，我们检测了缺氧/复氧（H/R）对TRAIL细胞毒性的影响。
利用H/R模型，将原代正常人肝内胆管上皮细胞暴露于缺氧环境1小时，然后进行复氧。检测死亡受体4（DR4）和DR5 mRNA及蛋白的表达。缺氧1小时后，用不同浓度的TRAIL处理胆管上皮细胞4小时。通过凋亡分析和甲基噻唑基四氮唑来确认胆管上皮细胞的死亡情况。采用荧光测定法测定半胱天冬酶-3和半胱天冬酶-8的活性。
与常氧培养的细胞相比，复氧后0分钟时DR4和DR5的mRNA表达上调，在复氧后60分钟达到峰值。DR4的蛋白表达在复氧后90分钟时最强；DR5的最强表达出现在复氧后120分钟。TRAIL处理组的凋亡率增加，TRAIL加H/R组的凋亡率进一步增加，且TRAIL介导的浓度依赖性细胞杀伤作用更明显。H/R后的半胱天冬酶-3和半胱天冬酶-8酶活性也随TRAIL浓度增加而增加。
H/R上调了DR4和DR5的表达，并增强了TRAIL介导的正常人肝内胆管上皮细胞凋亡。
