中文摘要：

目的研究铝诱导的神经母细胞瘤细胞的死亡方式及途径,探讨其毒理学及在神经细胞退行性变中的意义。方法用铝染毒神经母细胞瘤细胞株制作铝致神经细胞损伤模型,在铝和Nec-1处理后用光学显微镜观察细胞的生长状况,CCK-8法检测细胞的活力变化,流式细胞仪检测细胞的凋亡率、坏死率、线粒体膜电位和活性氧含量,电子显微镜观察细胞的超微结构变化。蛋白质印迹（Western blot）法检测LC3蛋白表达量。结果铝染毒神经母细胞瘤细胞形态学观察结果表明,凋亡与坏死并存于染铝细胞中;流式细胞术定量检测铝染毒后细胞的凋亡率和坏死率均显著升高（P〈0.05,P〈0.01）。超微结构显示有凋亡早期和晚期典型的核凝聚和多量典型双层膜结构的自噬小体。铝染毒SH-SY5Y细胞中加入Nec-1后,可以有效地降低细胞的坏死率（P〈0.05,P〈0.01）,显著降低线粒体的膜电位（P〈0.05,P〈0.01）,有效减少自噬体的形成及发展,且与细胞的自噬过程有关。Western blot结果显示LC3蛋白表达量随Nec-1（0~90μmol/L）作用浓度的提高而显著下降（P〈0.05,P〈0.01）。结论铝诱导的神经母细胞瘤细胞的死亡方式不仅包括传统的凋亡和坏死,自噬和程序性坏死也存在于其中,这可能与铝致神经退行性变的细胞丢失方式有关。

英文摘要：

Objective To investigate the neuroblastoma cell death modes induced by aluminum and clarify the toxicological and pathogenesis significance of them. Methods Neural cell death model was established in neuroblastoma cell line by aluminum treatment. After the treatment of aluminum and Nec-1, light microscope was used to observe the cell status; cell viability was assayed by CCK-8 kit ; aggregation and condensation of nuclei were viewed under fluorescent microscope. Cytometry was performed to quantify the apoptotic rate, necrotic rate, mitochondrial membrane potential and reactive oxygen species. Electron microscopy was used to observe the uhrastructures of ceils, and immunocytochemical assay was carried out to observe the expressions of apoptotic related proteins. Results The outcomes of various experiments showed coexistent characteristics of apoptosis, necrosis and autophagy in aluminum-treated neuroblastoma cells as follows： cell swelling companied cell shrinkage, increment of apoptotic ceils and necrotic cells stained by AO-EB; elevations of apoptotic rate, necrotic rate, and mitochondrial membrane potential assayed by flow cytometry; nuclei aggregation and double membrane autophagosomes under electron microscope（ P 〈 0.05, P 〈 0.01 ）. Effects of Nec-1 on neuroblastoma cells treated by 4 mmol/L demonstrated that Nec-1 could decrease necrotic rate significantly（ P 〈 0.05, P 〈 0.01 ）, Nec-1 could decline mitochondria membrane potential significantly （ P 〈 0.05, P 〈 0.01 ）, Electron microscopy showed the decrease of autophagy formation and development. Bands of western blotting displayed that LC3 expression enhanced with aluminum concentration（ P 〈 0.05）, but decreased with increment of Nec-I （0 - 90 μmol/L） concentration（ P 〈 0.05, P 〈 0.01 ）. Conclusion Modes of cell death in neuroblastoma cells induced by aluminum include not only apoptosis and necrosis, but also autophagy and necroptosis, which may be related with neural cell loss induced by aluminum.