PC12细胞转染人GPx-1基因后的抗氧化损伤作用

摘要

Objective To study the protective effect and molecular mechanism of glutathione peroxidase 1 (GPx-1) high expression on PC12 cell damage mediated by hydrogen peroxide (H2O2). Methods PC12 cells were transfected with plncx plasmid containing human GPx-1 gene and plncx plasmid, and a PC12 cell line was established that could stably express human GPx-1 gene and plncx plasmid through continuous screening. Two means of intervention, H2O2 and sodium selenite + H2O2, were given to PC12, GPx-1-PC12 and plncx-PC12 cells, respectively. GPx activity of cells in each group was detected with GPx activity detection kit; the survival and apoptosis of cells were detected by MTT method and flow cytometer, respectively. The expression of NF-κBp65 was detected by immunohistochemistry technique. Results GPx activity of GPx-PC12 group cells was obviously higher than that of controls. Intervention of H2O2 and sodium selenite + H2O2 did not significantly affect the cell survival rate in PC12 and plncx-PC12 groups (P>0. 05). The cell survival rate was higher in GPx-1-PC12 group than in PC12 and plncx-PC12 groups (P<0.01); the early and late apoptosis rates were obviously lower in GPx-1-PC12 group than in PC12 and plncx-PC12 groups (P<0.01). The positive rate of NF-κBp65 protein decreased significantly in GPx-1-PC12 group compared with that in PC12 group (P<0. 01). Conclusion The overexpression of GPx-1 gene has a great protective effect on the oxidative damage of PC12 cell mediated by H2O2. GPx-1 can inhibit the occurrence of PC12 cell apoptosis and NF-ΚB expression during oxidative damage.%目的 探讨GPx-1高表达在H2O2介导的PC12细胞损伤时的保护作用及分子机制.方法 将PC12细胞转染含人GPx-1基因的plncx质粒及空载体plncx质粒,经持续筛选建立起稳定表达人GPx-1基因和空质粒plncx的PC12细胞系.对PC12、GPx-1-PC12、plncx-PC12 3组细胞,分别给予H2O2和亚硒酸钠+ H2O2干预方式,GPx活性检测试剂盒检测各组细胞GPx活性;MTT法检测细胞的存活情况;流式细胞仪检测细胞凋亡发生的情况;免疫细胞化学法观察NF-κBp65的表达情况.结果 GPx-1-PC12组细胞GPx活性较对照组明显增高;3组细胞分别给予2种干预后,PC12与plncx-PC12组细胞存活率比较,差异无统计学意义(P>0.05),GPx-1-PC12较PC12、plncx-PC12组细胞存活率明显增高(P<0.01);GPx-1-PC12较PC12、plncx-PC12细胞早期和晚期凋亡率明显下降,存在统计学差异(P<0.01);GPx-1-PC12较PC12细胞NF-κBp65表达明显减少(P<0.01).结论 GPx-1基因高表达对H2O2所致的PC12细胞氧化损伤有很好的保护作用;GPx-1可抑制PC12细胞氧化损伤时凋亡的发生及NF-κB的表达.