Wang Guqi, Gong Yuewen, Anderson Judy, Sun Dongfeng, Minuk Gerald, Roberts Michael S, Burczynski Frank J
Faculty of Pharmacy, University of Manitoba, Winnipeg, MB, Canada.
Hepatology. 2005 Oct;42(4):871-9. doi: 10.1002/hep.20857.
Liver fatty acid binding protein (L-FABP) contains amino acids that are known to possess antioxidant function. In this study, we tested the hypothesis that L-FABP may serve as an effective endogenous cytoprotectant against oxidative stress. Chang liver cells were selected as the experimental model because of their undetectable L-FABP mRNA level. Full-length L-FABP cDNA was subcloned into the mammalian expression vector pcDNA3.1 (pcDNA-FABP). Chang cells were stably transfected with pc-DNA-FABP or vector (pcDNA3.1) alone. Oxidative stress was induced by incubating cells with 400 micromol/L H2O2 or by subjecting cells to hypoxia/reoxygenation. Total cellular reactive oxygen species (ROS) was determined using the fluorescent probe DCF. Cellular damage induced by hypoxia/reoxygenation was assayed by lactate dehydrogenase (LDH) release. Expression of L-FABP was documented by regular reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, and Western blot. The pcDNA-FABP-transfected cells expressed full-length L-FABP mRNA, which was absent from vector-transfected control cells. Western blot showed expression of 14-kd L-FABP protein in pcDNA-FABP-transfected cells, but not in vector-transfected cells. Transfected cells showed decreased DCF fluorescence intensity under oxidative stress (H2O2 and hypoxia/reoxygenation) conditions versus control in inverse proportion to the level of L-FABP expression. Lower LDH release was observed in the higher L-FABP-expressed cells in hypoxia/reoxygenation experiments. In conclusion, we successfully transfected and cloned a Chang liver cell line that expressed the L-FABP gene. The L-FABP-expressing cell line had a reduced intracellular ROS level versus control. This finding implies that L-FABP has a significant role in oxidative stress.
肝脏脂肪酸结合蛋白（L-FABP）含有已知具有抗氧化功能的氨基酸。在本研究中，我们测试了L-FABP可能作为一种有效的内源性细胞保护剂抵御氧化应激的假说。选择Chang肝细胞作为实验模型，因为其L-FABP mRNA水平检测不到。将全长L-FABP cDNA亚克隆到哺乳动物表达载体pcDNA3.1（pcDNA-FABP）中。Chang细胞用pc-DNA-FABP或单独的载体（pcDNA3.1）进行稳定转染。通过用400微摩尔/升过氧化氢孵育细胞或使细胞经历缺氧/复氧来诱导氧化应激。使用荧光探针DCF测定细胞内活性氧（ROS）总量。通过乳酸脱氢酶（LDH）释放测定缺氧/复氧诱导的细胞损伤。通过常规逆转录聚合酶链反应（RT-PCR）、实时RT-PCR和蛋白质印迹法记录L-FABP的表达。pcDNA-FABP转染的细胞表达全长L-FABP mRNA，而载体转染的对照细胞中不存在该mRNA。蛋白质印迹显示在pcDNA-FABP转染的细胞中表达14-kd的L-FABP蛋白，而在载体转染的细胞中不表达。与对照相比，转染的细胞在氧化应激（过氧化氢和缺氧/复氧）条件下显示出降低的DCF荧光强度，与L-FABP表达水平成反比。在缺氧/复氧实验中，在L-FABP高表达的细胞中观察到较低的LDH释放。总之，我们成功转染并克隆了表达L-FABP基因的Chang肝细胞系。与对照相比，表达L-FABP的细胞系细胞内ROS水平降低。这一发现表明L-FABP在氧化应激中具有重要作用。
