DJ-1对MPP^+诱导的多巴胺能神经元损伤的保护机制研究

Protective mechanism of DJ-1 on MPP^+ induced PD dopaminergic neurons damage

目的探讨DJ-1基因对MPP+诱导的多巴胺能神经元损伤模型的保护机制。方法用神经生长因子(NGF)将PC12诱导成神经元样分化的细胞株(多巴胺能神经元)。用1-甲基-4苯基吡啶离子(MPP+)诱导帕金森病PC12细胞损伤模型,用CCK-8试剂盒检测细胞增殖活性,DHE染色法检测ROS水平变化。Western blot检测DJ-1和TH蛋白表达变化,RT-q PCR检测α-synuclein和凋亡相关基因的RNA水平。用p CDH1-cmv载体过表达DJ-1后检测DJ-1对MPP+环境中细胞的保护作用。用RT-q PCR检测α-synuclein、p53和Bax的表达变化。结果 160μmol/L的MPP+处理18 h后,PC12细胞活性降低,细胞内ROS水平升高,处理48 h后细胞凋亡增加。DJ-1和TH蛋白表达均明显下调,RNA水平上α-synuclein、p53和Bax表达增加。过表达DJ-1能够保护MPP+中的细胞活性,抑制ROS水平升高。α-synuclein以及凋亡基因p53和Bax的表达升高受到抑制,细胞凋亡减少。结论 MPP+作用于多巴胺神经元,可以下调DJ-1和TH蛋白,促进α-synuclein积累ROS水平升高,并使p53和Bax表达增加,导致细胞凋亡增加。DJ-1基因能够能够在MPP+处理时抑制p53和Bax的表达,减少α-synuclein积累,降低细胞内ROS水平,保护细胞免受MPP+引起的损伤。

Objective To investigate the protective mechanism of DJ-1 on 1-methy-4-phenylpyridinium（MPP＋） induced Parkinson＇s disease（PD） PC12 dopaminergic neurons cell damage. Methods PC12 cells were induced into neurons differentiated cells（dopaminergic neurons） using nerve growth factor（NGF）. MPP＋was applied to induce PC12 cell injury model of PD. Cell activity was detected by CCK-8 cell proliferation kit. The change of reactive oxygen species（ROS） levels was monitored by using dihydro-ethidium（DHE）. The expression levels of DJ-1 and TH protein were detected by Western blot. The RNA level of α-synuclein and apoptosis related genes（p53 and Bax） were detected by RT-q PCR. DJ-1 expressing vector was constructed and transfected into PC12 cells, MPP＋was used to induce dopaminergic neuron injury model. Results After processed with 160 μmol/L MPP＋for 18 h, PC12 cell viability was decreased, and the level of endogenous ROS was increased. After processed with MPP＋for 48 h, apoptosis was increased, the expression of DJ-1 and TH protein were significantly decreased, and RNA level of α-synuclein, Bax and p53 were all increased. In DJ-1 overexpression cells, the decrease of cell viability and apoptosis induced by MPP＋were weaken, the RNA level of α-synuclein, p53, Bax and caspase apoptosis didn＇t emerge an obvious increasing any more.Conclusion MPP＋can cause the down-regulation of DJ-1 and TH protein. In addition, MPP＋up-regulate the expression of α-synuclein, Bax and p53, which led to cell apoptosis. DJ-1 can suppress the change of α-synuclein, Bax and p53, reduce accumulation of α-synuclein, and protect cells from MPP＋induced damage.