低剂量纳米银暴露致小鼠巨噬细胞RAW264.7的应激反应  

Stress response of mice macrophage RAW264.7 cell exposed to low dose of silver nanoparticles

作　　者：赵琳[1,2] 陈瑞[2] 龙鼎新[1] 陈锋[1] 陈春英[2] 

机构地区：[1]南华大学公共卫生学院,湖南衡阳421001 [2]国家纳米科学中心中国科学院纳米生物效应与安全性重点实验室,北京100190

出　　处：《中国当代医药》2015年第13期16-21,共6页China Modern Medicine

基　　金：国家自然科学基金(21477029)

摘　　要：目的探讨纳米银(AgNPs)对小鼠巨噬细胞RAW264.7毒性效应的影响,为AgNPs生物安全性的研究提供依据。方法将不同浓度的AgNPs(0、6.25、12.5、25、50、60、80μg/ml)和RAW264.7共同培养,8、24 h后采用甲基噻唑基四唑比色法(MTT)测定各组细胞的活力,选取2.5、5μg/ml两个无明显细胞毒性的浓度进行后续研究,检测应激相关基因和蛋白表达的变化;采用酶联免疫吸附试验(ELISA)检测细胞培养液中炎症因子TNF-α和IL-6的表达。结果用2.5、5μg/ml的AgNPs处理细胞,24 h后相差显微镜下可见Ag NPs进入细胞内,且5μg/ml组的细胞形态有明显改变;8、24 h后ELISA结果显示,IL-6无明显变化,在5μg/ml的24 h处理组TNF-α表达有显著升高,内质网应激相关的基因和蛋白在5μg/ml的24 h处理组表达明显上调。结论 RAW264.7细胞暴露于低剂量的AgNPs后表现出明显的应激反应,并且与暴露剂量和作用时间呈正相关。低剂量AgNPs能够干扰细胞的正常生理功能,长期暴露可能产生不可逆的损伤,应引起高度关注。Objective To investigate the toxicity of silver nanoparticles （AgNPs）on mice macrophage cell line RAW264.7 and provide the data for understanding their biosafety. Methods Cell viability was determined by the MTT method in RAW264.7 cells after incubation with AgNPs（0,6.25,12.5,25,50,60,80μg/ml）.The dose of 2.5,5μg/ml did not cause significant cytotoxicity to RAW264.7 cells,which were used for the following cell treatment to observe the cellular morphology and to determine the protein expression of the stress-related biomarker.The level of TNF-α and IL-6 in the culture medium were determined by ELISA. Results When the RAW264.7 cells were treated with 2.5 and 5 μg/ml of AgNPs for 24-h,the AgNPs were found to be internalized into the cells and cellular morphology were changed appar-ently in the 5 μg/ml group.The expression of IL-6 did not change after AgNPs treatement for 8 and 24 hours,but the expression of TNF-α was increased in the group after AgNPs treatement for 24-h.Further,the endoplasmic reticulum stress response maker and oxidative-stress protein were upregulated significantly in RAW264.7 cells after 5 μg/ml Ag-NPs treatement for 24-h. Conclusion RAW264.7 cells after exposure to low dose of AgNPs show obvious stress reac-tion,which is positively correlated with exposure dose and effect time.Low dose AgNPs exposure can disturb the normal cellular physiological function on RAW264.7 cells.Prolonged exposure may cause irreversible damage, should arouse at-tention.

关 键 词：纳米银 低剂量 毒性 内质网应激 

分 类 号：R122[医药卫生—环境卫生学]