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目的:研究纳米二氧化硅(SiO2)颗粒的血管内皮细胞毒性和可能的作用机制,为探讨纳米SiO2颗粒毒性效应及安全性评价提供参考依据。方法:将体外培养的人脐静脉内皮细胞(HUVECs)随机分为对照组和不同浓度纳米SiO2颗粒暴露组,暴露浓度分别为12.5、25.0、50.0及100.0 mg·L-1。采用透射电镜(TEM)观察纳米SiO2颗粒粒径、形貌及分散性。细胞处理24h后,电感耦合等离子体原子发射光谱(ICPAES)法测定细胞中硅水平;MTT法测定细胞活力;乳酸脱氢酶(LDH)释放法检测细胞膜的完整性;DCFHDA荧光探针标记激光共聚焦显微镜观察细胞中活性氧(ROS)水平;DAPI染色荧光显微镜下观察细胞核形态;AnnexinⅤ/PI双染标记流式细胞术(FCM)检测细胞凋亡率;罗丹明123线粒体试剂盒检测细胞线粒体膜电位。结果:TEM观察,纳米SiO2颗粒分布均匀,大小一致,颗粒呈球形,分散性好,未发生聚集。用Image J软件计算得到颗粒平均粒径为(57.66±7.30)nm。与对照组比较,纳米SiO2颗粒作用于HUVECs后,细胞活力下降(P<0.05),细胞中硅水平和培养液中LDH活性增加(P<0.05),细胞中ROS水平升高,线粒体膜电位下降,甚至发生细胞凋亡。上述细胞效应均表现为随颗粒作用浓度的增加而逐渐明显,呈现一定的剂量依赖关系。结论:纳米SiO2颗粒具有血管内皮细胞毒性,可诱导ROS生成和氧化应激,从而破坏细胞膜、损伤线粒体,最终引发细胞凋亡。
OBJECTIVE: To study the vascular endothelial cell toxicity and possible mechanism of nano-silica (SiO2) particles and to provide a reference for the evaluation of the toxicity and safety of nano-SiO2 particles. Methods: Human umbilical vein endothelial cells (HUVECs) cultured in vitro were randomly divided into control group and exposed groups with different concentrations of nano-SiO2 particles. The exposed concentrations were 12.5, 25.0, 50.0 and 100.0 mg · L-1, respectively. Transmission electron microscopy (TEM) was used to observe the particle size, morphology and dispersion of nano-SiO2 particles. The cells were treated with ICPAES for 24 hours. The cell viability was measured by MTT assay and the cell membrane integrity by lactate dehydrogenase (LDH) release assay. The DCFHDA fluorescent labeled laser The level of reactive oxygen species (ROS) in the cells was observed by focusing microscope. The morphology of nucleus was observed under DAPI staining by fluorescence microscopy. The apoptosis rate was detected by AnnexinⅤ / PI double staining flow cytometry (FCM). The mitochondrial membrane Potential. Results: TEM observation showed that the nano-SiO2 particles were uniformly distributed, uniform in size and spherical in shape, with good dispersibility and no aggregation. The average diameter of the particles calculated by Image J software was (57.66 ± 7.30) nm. Compared with the control group, the cell viability was decreased (P <0.05), the level of silicon in the cells and the activity of LDH in the culture medium were increased (P <0.05), the level of ROS in the cells was increased and the mitochondrial membrane potential was decreased , And even apoptosis occurred. The above-mentioned cellular effects are shown as the concentration of the particles increases and gradually became apparent, showing a certain dose-dependent relationship. Conclusion: Nano-SiO2 particles have vascular endothelial cell toxicity, which can induce ROS generation and oxidative stress, thus damaging the cell membrane and damaging mitochondria, eventually leading to apoptosis.