DJ-1对MPP+诱导的多巴胺能神经元损伤的保护机制研究

摘要

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-qPCR检测α-synuclein和凋亡相关基因的RNA水平.用pCDH1-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-qPCR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 in-duced into neurons differentiated cells (dopaminergic neurons) using nerve growth factor (NGF). MPP+was applied to in-duce 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 ofα-synuclein and apoptosis related genes (p53 and Bax) were de-tected by RT-qPCR. DJ-1 expressing vector was constructed and transfected into PC12 cells, MPP+was used to induce dopaminergic neuron injury model. Results After processed with 160μmol/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 in-creased, the expression of DJ-1 and TH protein were significantly decreased, and RNA level ofα-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 ofα-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 expres-sion ofα-synuclein, Bax and p53, which led to cell apoptosis. DJ-1 can suppress the change ofα-synuclein, Bax and p53, reduce accumulation ofα-synuclein, and protect cells from MPP+induced damage.