野生型DJ-1基因在体外培养SH-SY5Y细胞中高表达对氧化损伤的抑制作用

The inhibitive effects of over-expression of DJ-1 gene on oxidative injury in SH-SY5Y cells in vitro

目的:研究帕金森病相关基因野生型DJ-1高表达对鱼藤酮及过氧化氢致多巴胺神经元SH-SY5Y细胞氧化损伤的保护作用及机制。方法:将pcDNA3-FLAG-DJ-1质粒转染进入SH-SY5Y细胞,采用Western blot分析观察DJ-1在细胞内的表达,以2,7-二氯荧光黄双乙酸盐(DCFH-DA)作为探针,用流式细胞仪检测鱼藤酮诱导的活性氧水平,以MTT法检测过氧化氢损伤的细胞存活率变化。结果:野生型DJ-1被转染进入SH-SY5Y细胞中,并能高表达。野生型DJ-1的高表达可减少活性氧的生成,0.5μmo.lL-1浓度的鱼藤酮处理细胞48 h后,细胞内存在大量活性氧,M2期细胞明显减少(转染野生型DJ-1的SH-SY5Y细胞71.60%与未转染DJ-1的SH-SY5Y细胞89.19%相比)。并且可以抑制过氧化氢导致的细胞存活率下降,过氧化氢处理1 h后,野生型DJ-1转染的细胞存活率明显高于未转染者,过氧化氢浓度为0.1,0.2和0.5 mmol.L-1,细胞存活率分别由77.71%,66.28%和39.08%上升至93.01%,83.96%和60.15%。结论:野生型DJ-1基因在体外培养SH-SY5Y细胞中高表达对氧化损伤具有抑制作用,该作用可能与降低细胞内活性氧水平有关。

Objective： To investigate the anti-oxidative effects of Parkinson＇s disease-related wild-type （WT） DJ-1 gene in SH-SYSY cells in vitro. Methods： Plasmid pcDNA3-FLAG-DJ-1 was transfected into SH-SY5Y cells followed by Western blot analysis for detecting DJ-1 expression. Flow cytometric assay was performed to determine the level of reactive oxygen species （ROS） after reaction with DCFH-DA, and MTT assay was used to determine the cell viability. Results： The result of Western blot analysis showed that the D J-1 gene was over-expressed in transfected SH-SYSY cells. Overvexpression of WT DJ-1 gene reduced the levels of ROS induced by rotenone. The number of M2 phase cells after treated with 0.5μmol·L ^-1 rotenone for 48 h was lower in transfected cells than in native cells （71.60% vs 89.19% ）. In addition, over-expression of WT DJ-1 gene inhibited the reduction of cell viability induced by H202. After cells were treated with 0.1, 0.2 and 0.5 mmol·L^-1 H202 for 1 h, the cell viabilities of transfected cells （93.01% , 83.96% and 60.15% , respectively） were significantly higher than those of native cells （77.71% , 66.28% and 39.08% , respectively）. Conclusion： Over-expression of WT DJ- 1 gene can protect SH-SY5Y cells from oxidative stress induced by rotenone or H202. This protective effect of WT DJ-1 may relate to reduction of ROS.