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目的探讨多巴胺(DA)神经元诱变剂对细胞内泛素化α-synuclein聚集的影响及其细胞损伤作用。方法应用MPP~+、特异性泛素-蛋白酶体系统(UPS)抑制剂lactacystin及H_2O_2处理神经生长因子(NGF)诱导的PC12细胞株与N2a细胞株16h后,采用免疫荧光双标记的方法在共聚焦显微镜下观察细胞内泛素化α-synuclein聚集,比较三种诱变剂对这两种细胞株作用的差异。在PC12细胞中加入不同终浓度的lactacystin处理24h,MTT方法检测PC12细胞活力。10μmol/L lactacystin处理PC12细胞不同时间,流式细胞术检测PC12细胞的早期凋亡率。结果经三种诱变剂处理后,MPP~+和lactacystin选择性地诱发PC12细胞内泛素化α-synuclein聚集且以lactacystin的作用更为显著,N2a细胞则无明显的泛素化α-synuclein聚集。H_2O_2作用于PC12细胞的效应与MPP~+相近,但仅引起N2a细胞内少量泛素化α-synuclein聚集。经lactacystin处理后的PC12细胞活力呈剂量依赖性下降;5μmol/L、10μmol/L和20μmol/L lactacystin处理24h后细胞存活率分别为79.5%±2.1%、49.3%±3.2%和31.2%±2.8%(与对照组相比,均P<0.01)。流式细胞术显示lactacystin处理后PC12细胞早期出现凋亡细胞,并且随着处理时间的延长,细胞凋亡率逐渐增加。结论特异性UPS和线粒体呼吸链抑制剂选择性作用于DA神经元,诱导细胞内泛素化α-synuclein聚集,终使细胞发生凋亡,而以氧化应激为主要损伤途径的诱变剂的作用则不具有选择性。
Objective To investigate the effect of dopamine (DA) neuron mutagens on the aggregation of intracellular ubiquitinated α-synuclein and its cell injury. Methods PC12 cells and N2a cells induced by MPP ~ +, lactacystin and H_2O_2, a specific inhibitor of ubiquitin-proteasome system (UPS), were treated with NGF for 16 hours. The immunofluorescence double labeling Under the microscope, we observed the aggregation of ubiquitinated α-synuclein in cells and compared the effects of three mutagens on these two cell lines. The PC12 cells were treated with different final concentrations of lactacystin for 24 hours. The viability of PC12 cells was detected by MTT assay. PC12 cells were treated with 10μmol / L lactacystin for different time. The early apoptosis rate of PC12 cells was detected by flow cytometry. Results MPP ~ + and lactacystin selectively induced the aggregation of ubiquitinated α-synuclein in PC12 cells with lactacystin, but not with ubiquitinated α-synuclein Gather together. The effect of H_2O_2 on PC12 cells was similar to that of MPP ~ +, but only a small amount of ubiquitinated α-synuclein was accumulated in N2a cells. The viability of PC12 cells treated with lactacystin decreased in a dose-dependent manner. The viability of PC12 cells treated with lactacystin at 5μmol / L, 10μmol / L and 20μmol / L for 24 h were 79.5% ± 2.1%, 49.3% ± 3.2% and 31.2% ± 2.8, respectively % (All P <0.01 compared with the control group). Flow cytometry showed that PC12 cells appeared apoptotic cells early after lactacystin treatment, and the apoptosis rate increased gradually with the prolonging of treatment time. Conclusion Specific UPS and mitochondrial respiratory chain inhibitors act selectively on DA neurons, induce the aggregation of ubiquitinated α-synuclein in cells, and finally induce the apoptosis of cells. However, mutants with oxidative stress as the main pathways of injury The role is not selective.