中文摘要：

目的探讨不同粒径二氧化硅（Si O_2）颗粒诱导的细胞自噬,进一步阐明其机制。方法以体外培养的支气管上皮细胞（BEAS-2B）为模型,随机分为对照组和Si O_2（Si200,Nano-Si60,Nano-Si40）颗粒暴露组,暴露浓度为25μg/ml。采用透射电子显微镜（TEM）观察Si O_2粒径、形貌及分散性。细胞处理24 h后,MTT法测定细胞活力;透射电子显微镜观察Si O_2颗粒摄取及细胞自噬超微结构;Western blot检测3-MA预处理后自噬标志蛋白LC3的表达。结果电镜显示3种Si O_2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分别为46.26±5.68 nm、61.51±7.82 nm和206.31±6.35 nm。与对照组相比,Si O_2颗粒作用于BEAS-2B细胞后,细胞活力下降（P〈0.05）;电子显微镜观察显示Nano-Si60和Nano-Si40处理组细胞内呈现出自噬泡累积,自噬泡内含有明显的高电子密度Si O_2颗粒及被部分降解的细胞内物质并伴有不同程度的线粒体损伤;Western blot显示LC3蛋白水平在Nano-Si60和Nano-Si40处理组有明显变化,加入3-MA预处理后LC3表达受到抑制。结论 Si O_2颗粒可降低支气管上皮细胞存活率,并具有剂量和粒径依赖趋势;纳米级Si O_2颗能够诱导细胞发生自噬而微米级Si O_2颗粒则不诱导细胞自噬;3-MA能够在一定程度上抑制Si O_2颗粒诱导细胞自噬发生。

英文摘要：

Objective To investigate the autophagy induced by different sizes of silica particles（ Si200,Nano-Si60,Nano-Si40） on cells,and to further clarify the mechanisms of autophagy induced by silica particles. Methods The cultured Bronchial epithelial cells（ BEAS-2B） were used as cell model in vitro,and randomly divided into control and silica particles（ Si200,Nano-Si60,Nano-Si40） exposure groups at the concentrations of 25 μg / ml respectively. Transmission electron microscope（ TEM） was used to determine particle size,morphology and dispersion of silica particles. After 24 h exposure,MTT assay was the determination of cell viability. TEM was employed to detect whether cellular uptake and autophagy was induced by silica particles. Western blot was utilized to detect the expression of autophagy marker protein of LC3. Results The TEM images showed that silica particles were mostly spherical with uniform size and well dispersed.The average particle size were 46. 26 ± 5. 68 nm,61. 51 ± 7. 82 nm,206. 31 ± 6. 35 nm calculated by image J software. Compared with the control group,cell viability in silica particles exposure groups was inhibited（ P 0. 05）. Electron microscope showed that typical autophagic vacuoles with degraded cytoplasmic contents and highly electron-dense silica particles in BEAS-2B cells, with varying degrees of mitochondrial damage in Nano-Si60 and Nano-Si40 treatment groups. Western blot demonstrated that the level of LC3-II protein gradually increased in Nano-Si60 and Nano-Si40 treatment groups,after adding 3-MA pretreatment LC3 expression was suppressed. Conclusion It was indicated that silica particles induced cytotoxicity in dose and size-dependent manner. Nano silica particles can cause autophagy BEAS-2B cells in vitro,but micron grade silica particles cannot induce autophagy. 3-MA can inhibit cell autophagy induced by silica particles in a certain extent.