摘要
目的探讨1-甲基-4-苯基吡啶离子(MPP+)诱导线粒体自噬在帕金森病(PD)发病机制中的作用。方法将细胞分为MPP+(0 mmol/L)对照组、MPP+(1 mmol/L)处理组和MPP+(2 mmol/L)处理组,共同转染EGFP-LC3和RFP-MI-TO后加入MPP+处理48 h。Western blot检测细胞自噬水平的变化,甲丹磺酸尸胺(MDC)检测自噬空泡聚集,免疫荧光法检测EGFP-LC3和RFP-MITO亚细胞共定位,流式技术检测线粒体膜电位及活性氧。结果 MPP+1 mmol/L及MPP+2 mmol/L组LAMP2 A、Beclin1和LC3-Ⅱ/LC3-Ⅰ的灰度值与对照组相比均上调,差异有统计学意义(P<0.05)。与对照组相比,MPP+处理组自噬水平增加,自噬空泡增加,外源性LC3表达上调,EGFP-LC3和RFP-MITO存在亚细胞共定位。MPP+1 mmol/L及MPP+2 mmol/L组线粒体膜电位较对照组降低;MPP+1 mmol/L及MPP+2 mmol/L组线粒体活性氧较对照组增加,差异均有统计学意义(P<0.05)。结论 MPP+通过调控线粒体自噬水平致线粒体氧化应激损伤。
Objective To investigate the role of 1-methyl-4-pehny1-pyridine(MPP+) regulated mitochondrial autophagy in the pathogenesis of Parkinson's disease.Methods Human neuroblastoma cell lines(SH-SY5Y) were treated with MPP+(0,1 or 2 mmol/L) for 48 hrs and then were transfected with EGFP-LC3 and RFP-MITO.Expression of LAMP2A,Beclin1 and LC3-II/LC-I proteins in SH-SY5Y cells was detected by Western blot after MPP+ treatment for 48 hrs.Cellular immunefluorescent chemical method was used to observe the change of autophagic vacuolization and the signals of EGFP-LC3 and RFP-MITO,and their subcellular colocalization.The flow cytometry was used to detect the mitochondrial membrane potential and reactive oxygen species content.Results The gray values of LAMP2A,Beclin1 and LC3-II/LC3-I in the 1 mmol/L and 2 mmol/L MPP+ treatment groups were significantly increased compared with the control group(0 mmol/L MPP+ treat-ment)(P〈0.05).The autophagy levels increased,the number of autophagic vacuolization increased,and expression of exogenous LC3 increased significantly in the two MPP+ treatment groups compared with the control group.EGFP-LC3 and RFP-MITO subcellular colocalization was observed in the MPP+ treatment groups.Mitochondrial reactive oxygen increased and mitochondrial membrane potential decreased significantly in the two MPP+ treatment groups compared with the control group.Conclusions MPP+ can induce mitochondrial oxidative stress injury through regulating levels of mitochondrial autophagy.
出处
《国际神经病学神经外科学杂志》
2012年第6期489-493,共5页
Journal of International Neurology and Neurosurgery
基金
国家自然科学基金(81241046
81100949)