论文部分内容阅读
目的研究纳米氧化铝对新生Wistar乳大鼠皮质神经元线粒体自噬的影响。方法神经元原代培养4 d进行纯度鉴定,13 nm纳米氧化铝0.5 mmol·L-1染毒12,24和48 h,检测培养液上清中乳酸脱氢酶(LDH)活性;流式细胞仪检测线粒体膜电位(MMP);透射电镜观察线粒体的超微结构和线粒体自噬体;单丹酰戊二胺(MDC)荧光染色观察自噬小体的形成;Western蛋白质印迹法检测Bcl-2结合蛋白1(Beclin1)和微管相关蛋白1轻链3(LC3)Ⅱ/Ⅰ蛋白的表达;溶酶体红色荧光探针Lysotracker和线粒体绿色荧光探针Mitotracker共染色观察溶酶体与线粒体的融合。结果神经元培养4 d,经纯度鉴定95%以上为神经元。与正常对照组相比,纳米氧化铝0.5 mmol·L-1染毒12和24 h组上清液中LDH活性均明显增加(P<0.05),但随着染毒时间的延长LDH活性呈下降趋势。MMP测定结果显示,随着染毒时间的延长,MMP逐渐降低(P<0.01)。细胞超微结构观察发现,纳米氧化铝组有线粒体肿胀和空泡形成,并可检测到线粒体自噬体。MDC荧光染色显示,正常对照组仅见极少量的MDC阳性荧光颗粒,纳米氧化铝组可见明显的MDC阳性荧光颗粒。自噬相关蛋白Beclin1和LC3Ⅱ/Ⅰ的表达随着染毒时间的延长逐渐增加(P<0.05)。荧光共染色显示,纳米氧化铝组线粒体与溶酶体融合。结论纳米氧化铝可引起神经元发生自噬以及线粒体损伤,受损的线粒体可能通过线粒体自噬作用清除。
Objective To investigate the effects of nano-alumina on mitochondrial autophagy in cortical neurons of newborn Wistar rats. Methods Primary culture of neurons was performed for 4 days for purity identification. 13 mmol Nano-alumina 0.5 mmol·L-1 was exposed to 12, 24 and 48 hours respectively to detect the activity of lactate dehydrogenase (LDH) in the culture supernatant. Flow cytometry The mitochondrial membrane potential (MMP) was detected by transmission electron microscopy. The ultrastructure of mitochondria and mitochondrial autophagosome were observed under transmission electron microscope. The formation of autophagy was observed by fluorodeoxyglucoside (MDC) staining. Western blotting was used to detect the expression of Bcl-2 (Beclin1) and light chain 3 (LC3) Ⅱ / Ⅰ protein of microtubule-associated protein 1. The lysosome-mitochondrial fusion was observed by lysosomal red fluorescent probe Lysotracker and mitochondria green fluorescent probe Mitotracker co-staining . Results The neurons were cultured for 4 days and more than 95% of the neurons were identified by their purity. Compared with the normal control group, the activity of LDH in the supernatant of 0.5 mmol·L-1 nano-alumina exposure group for 12 and 24 h increased significantly (P <0.05), but the activity of LDH decreased with the prolongation of exposure time trend. The results of MMP assay showed that MMP gradually decreased with the prolongation of exposure time (P <0.01). Ultrastructural observation revealed that mitochondria were swollen and vacuolated in the nano-alumina group, and mitochondrial autophagosomes could be detected. MDC fluorescence staining showed that only a very small amount of MDC-positive fluorescent particles were observed in the normal control group, and obvious MDC-positive fluorescent particles were observed in the nano-alumina group. The expression of autophagy-related proteins Beclin1 and LC3Ⅱ / Ⅰ increased with the prolongation of exposure time (P <0.05). Fluorescent staining showed that the nano alumina group mitochondria and lysosome fusion. Conclusion Nano-alumina can cause autophagy in neurons and mitochondrial damage, and damaged mitochondria may be cleared by mitophagy.