目的: 探讨氧化钕(Nd2O3)暴露对星形胶质细胞(AST)线粒体的损伤,以及隧道纳米管(TNTs)在其中的作用。方法: 采用新生3 d内的SD大鼠提取AST,通过免疫荧光鉴定胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)特异性表达的情况;CCK8试剂盒检测Nd2O3暴露后的细胞存活率;2,7-二氯荧光素二乙酸酯(2',7'-Dichlorodihydrofluorescein diacetate, DCFH-DA)探针检测细胞内活性氧;红色线粒体超氧化物荧光探针(MitoSOX Red Mitochondrial Superoxide Indicator, MitoSOX Red)检测线粒体内活性氧;罗丹明 123(Rhodamine 123)检测线粒体膜电位;活细胞染色标记F-肌动蛋白(F-actin)、β-微管蛋白(β-tubulin)以及线粒体观察TNTs结构、数量和线粒体的传递。结果: 提取的星形胶质细胞特异性表达GFAP;通过细胞活力检测确定最佳暴露剂量和时间分别为10 μg/mL Nd2O3和12 h;Nd2O3暴露后细胞内活性氧、线粒体内活性氧水平均明显增高,线粒体膜电位下降;AST间TNTs的结构以F-actin为主;Nd2O3暴露后AST间TNTs的数量增多;Nd2O3暴露后,AST间生成的TNTs促进线粒体的传递。结论: Nd2O3致AST氧化应激水平升高,引发线粒体损伤,并诱导AST间生成TNTs促进线粒体的传递。
Objective: To investigate the damage of neodymium oxide (Nd2O3) exposure on mitochondria of astrocytes (AST) and the role of tunneling nanotubes (TNTs) in it. Methods: AST was extracted from SD rats within 3 days of birth, and the specific expression of GFAP was identified by immunofluorescence. CCK8 kit was used to detect the cell survival rate after neodymium oxide exposure. DCFH-DA probe was used to detect intracellular reactive oxygen species; MitoSOX Red was used to detect the reactive oxygen species in mitochondria. Mitochondrial membrane potential was detected by Rhodamine 123. The live cell staining markers F-actin, β-tubulin and mitochondria were used to observe the structure, quantity and mitochondrial transmission of TNTs. Results: The extracted astrocytes specifically expressed GFAP; the optimal exposure dose and time were determined by cell viability testing to be 10 μg/mL Nd2O3 and 12 hours. After Nd2O3 exposure, the levels of intracellular reactive oxygen species and mitochondrial reactive oxygen species were significantly increased, and the mitochondrial membrane potential was decreased; the structure of TNTs in AST-AST was dominated by F-actin; and after Nd2O3 exposure, the number of TNTs in ASTs was increased, while TNTs mediated mitochondrial transmission in ASTs after Nd2O3 exposure. Conclusion: The increased level of oxidative stress in AST caused by Nd2O3 has been demonstrated to trigger mitochondrial damage and to mediate intercellular mitochondrial transmission via TNTs.
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