纳米二氧化硅对BEAS-2B细胞的毒性研究

In vitro toxicity of silica nanoparticles in BEAS-2B cells

目的评价纳米二氧化硅颗粒物对人正常支气管上皮细胞（BEAS-2B）的毒性作用及可能机制。方法（1）扫描电子显微镜（Scanning Electron Microscope,SEM）观察经100μg/ml的20 nm二氧化硅和结晶型二氧化硅（MinU-Sil 5）暴露24~72 h后的BEAS-2B细胞形态变化;（2）应用3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT）比色法检测实验组（纳米二氧化硅处理组）、阳性对照组（Min-U-Sil 5处理组）的细胞活力变化,分别测定不同实验条件下的还原型谷胱甘肽（GSH）、细胞内活性氧（ROS）和乳酸脱氢酶（LDH）水平;（3）流式细胞仪分析经暴露不同浓度20 nm二氧化硅后的细胞周期变化。结果 （1）经20 nm二氧化硅刺激24 h后BEAS-2B细胞体积增大,胞浆疏松,细胞数量减少,并随刺激时间延长而加重,阳性对照组上述变化不明显;（2）BEAS-2B细胞活力随暴露浓度和暴露时间的增加而下降,暴露浓度〉50μg/ml时20 nm二氧化硅组的细胞活力下降与阳性对照组间比较,差异有统计学意义（P〈0.05）,暴露浓度为100和200μg/ml时20 nm组与50 nm组间比较差异有统计学意义（P〈0.05）。100μg/ml浓度暴露24 h后,20 nm二氧化硅组细胞活力下降情况与阳性对照组间比较,差异有统计学意义（P〈0.05）,暴露72 h后各组间细胞活力下降差异均有统计学意义（P〈0.05）。BEAS-2B细胞培养基中的细胞内活性氧（ROS）升高、还原型谷胱甘肽（GSH）下降和乳酸脱氢酶（LDH）升高,并随暴露时间延长和二氧化硅粒径减小而变化;（3）纳米二氧化硅可使G2/M期停滞和G1期细胞增多,并呈现浓度效应。结论纳米二氧化硅造成BEAS-2B细胞的细胞毒性作用强于结晶型二氧化硅（Min-U-Sil 5）,纳米二氧化硅对BEAS-2B细胞的细胞毒性作用,具有浓度-效应、时间-效应和粒径-效应现象,细胞毒性作用并最终引起细胞氧化损伤。

Objective In order to elucidate the Silica nanoparticles-induced to the BEAS-2B cell cytotoxicity and its mechanism.Methods（ 1） The morphology of BEAS-2B cells after 24 ~ 72 h exposed to 20 nm Si O2 nanopaticles or crystalline Si O2（ Min-U-Sil 5）particles in a concentration of 100 μg / ml were observed by scanning electron microscope.（ 2） Cell viability of the experimental group（ Si O2nanoparticles） and its changes of positive group（ Min-U-Sil 5） were detected by the 3-（ 4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（ MTT） method,and the glutathione（ GSH）,reactive oxygen species（ ROS）,lactate dehydrogenase（ LDH） were also measured under various conditions.（ 3） The cell cycle changes exposed to 20 nm Si O2 nanopaticles in different dosage were analyzed by flow cytometer.Results（ 1） After 24 h exposure to Si O2,the BEAS-2B cells volu7 me increases,including pulp loose,reducing the number of cells and g aggravated with prolonged stimulation,and there was no significant changes in positive group.（ 2） The BEAS-2B cells viability decreased in a dose-dependent manner and time-dependent manner. Cell viability was significantly decreased in 20 nm Si O2 group compared to positive group in concentration of 50 μg / ml. The difference was statistically significant bewteen 20 nm and 50 nm group in concentration of 100μg / ml and 200 μg /ml respectively. There as also a significant decrease bewteen the 20 nm Si O2 group and positive group after 24 h exposure（ P〈0. 05）. And it showed the same difference in all groups after 72 h exposure（ P〈0. 05）. The intracellular reactive oxygen species（ ROS） increased,glutathione（ GSH） decreased and lactate dehydrogenase（ LDH） elevated of the BEAS-2B cell culture medium in a dosedependent manner and size-dependent manner.（ 3） The Si O2 nanoparticles can cause G2 / M phase arrest and G1 phase cells increased in a dose-dependent manner. Conclusions Exposure to Si O2 nanopaticles resulted in a time-dependent,size-dependent,and dose-dependent cytotoxicity in cultured BEAS-2B cells that was associated with increased oxidative stress.