论文部分内容阅读
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。
Objective To investigate the cell autophagy induced by particles of different particle size (Si O 2) and to elucidate its mechanism. Methods Bronchoalveolar epithelial cells (BEAS-2B) cultured in vitro were randomly divided into control group and exposure groups Si2O_2 (Si200, Nano-Si60, Nano-Si40). The exposure concentration was 25μg / ml. Transmission electron microscopy (TEM) was used to observe the particle size, morphology and dispersity of Si O_2. Cell viability was measured by MTT assay after 24 h of cell treatment. The ultrastructure of Si 2 O 2 particles was observed by transmission electron microscopy. The expression of autophagy marker LC3 was detected by Western blot. Results Electron microscopy showed that the three kinds of Si O 2 particles were uniformly distributed, uniform in size, spherical in shape and good in dispersibility without aggregation. The mean diameters of the particles calculated by Image J software were 46.26 ± 5.68 nm, 61.51 ± 7.82 nm and 206.31 ± 6.35 nm, respectively. Compared with the control group, Si O 2 particles decreased the viability of BEAS-2B cells (P <0.05); electron microscopy showed that Nano-Si60 and Nano-Si40 treated cells exhibited autophagic vacuoles, Which contained obvious high electron density Si O 2 particles and partly degraded intracellular substances accompanied by varying degrees of mitochondrial damage. Western blot showed that LC3 protein levels were significantly changed in the Nano-Si 60 and Nano-Si 40 treated groups, and 3- LC3 expression was inhibited after MA pretreatment. Conclusion Si O 2 particles can reduce the survival rate of bronchial epithelial cells in a dose- and size-dependent manner. Nanosized Si O 2 particles induce autophagy and micron-sized Si O 2 particles do not induce autophagy. 3-MA can activate To a certain extent, inhibition of Si O 2 particles induced cell autophagy.