不同粒径纳米二氧化硅的体外细胞膜毒性作用

Cell Membrane Injury Induced by Different Sizes of SiO_2 Nanoparticles in vitro

探讨不同粒径纳米二氧化硅对体外培养细胞膜的损伤作用及其机制。20nm、60nmSiO2及微米SiO2对RAW264.7巨噬细胞染毒后,分别采用MTT法、显微镜及透射电镜、能谱分析、荧光漂白恢复技术(FRAP)、丙酮酸法、荧光探针2′,7′-二乙酰二氯荧光素(DCFH-DA),测定染毒细胞的活性、形态及超微结构,细胞内外Si元素分布、膜流动性,细胞外液中乳酸脱氢酶(LDH)含量和细胞内活性氧生成。试验结果显示:20nm、60nmSiO2及微米SiO2粒子的细胞半数抑制浓度(IC50)分别为2.07、6.82和14.71mg/mL;染毒后细胞数量减少,呈气球样变,线粒体肿胀、破坏;细胞内吞噬泡和细胞间隙颗粒均含Si元素;31.25、125、500μg/mL的20nm、60nmSiO2组及500μg/mL微米SiO2组细胞膜荧光恢复率降低(P<0.05);125、500μg/mL两种粒径纳米SiO2组及500μg/mL微米SiO2组细胞膜扩散系数降低(P<0.05);两种粒径纳米SiO2组细胞外液中LDH活性升高(P<0.05),500μg/mL的20nmSiO2较60nmSiO2和微米SiO2致细胞荧光恢复率降低,LDH活性升高(P<0.05);两种粒径纳米SiO2及微米SiO2可致染毒细胞内活性氧水平升高(P<0.05)。结果表明,20nm、60nmSiO2可通过诱导脂质过氧化引起细胞生长抑制、形态异常、超微结构改变和细胞膜损伤,相同剂量20nmSiO2产生的毒性效应较60nmSiO2明显,说明纳米SiO2的尺寸效应是一个值得关注的问题。

The cell membrane injury and mechanism of 20 nm,60 nm SiO2 nanoparticles on RAW264.7 cells were evaluated in vitro.RAW264.7 cells were treated by 20 nm,60 nm SiO2 nanoparticles and micro SiO2 particles for 24 h.The viability of RAW264.7 cell was measured by MTT assay.The morphology and ultrastructural change were observed by light,fluorescence microscope and transmission electron microscopy.Membrane fluidity of the cells were measured through fluorescence recovery after photobleaching（ FRAP） technique with（ 2-（ 6-（ 7-nitrobenz-2-oxa-1,3-diazol-4-yl ） amino ） hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine）（NBD-C6-HPC） as the fluorescent probe.Lactate dehydrogenase（ LDH） in culture medium of RAW264.7 cells were assayed by pyruvic acid method.The content of reactive oxygen species（ ROS） in RAW264.7 cells were measured by fluorescence probe 2′,7′-chlorodihydrofluorescein diacetate（DCFH-DA）.The IC50 of 20-nm,60 nm SiO2 nanoparticles and micro SiO2 were 2.07,6.82 and 14.71 mg /mL,respectively.The cell number decreased,ballooning degeneration and mitochondria swelling after cells treated by three kinks of particles.Si elements were detected in lysosomes and intercellular spaces by energyspectrometer.The fluorescence recovery rate were lower than control（ P 0.05 ） after RAW264.7 cells exposured to 20 nm、60 nm SiO2 nanoparticles at 31.25,125,500 μg /mL and micro SiO2 at 500 μg /mL for 24 h.The diffusion coefficients of 20 nm,60 nm SiO2 nanoparticles at 125.00,500.00 μg /mL respectively and micro SiO2 at 500.00 μg /mL were lower than that of the control（ P 0.05）.The production of LDH was increased compared with the control（P 0.05）.The decrease of fluorescence recovery rate and the increase of LDH in 20 nm SiO2 nanoparticles group were much more obvious than 60 nm SiO2 nanoparticles and micro SiO2 groups.The level of ROS in RAW264.7 cells were increased obviously after treated by 20 nm,60 nm SiO2 nanoparticles and micro SiO2 particles（P 0.05）.Oxidative stress may be the mechanism of RAW264.7cell growth inhibition,morphological abnormality,ultrastructural change and membrane damage induced by 20 nm and 60 nm SiO2 nanoparticles.20 nm SiO2 nanoparticles were found to be more cytotoxicity than the 60 nm SiO2 nanoparticles.Different cytotoxicity effect induced by SiO2 nanoparticles was presented due to different size.The size effect is a noteworthy issue for the further toxicity research and standard formulation of SiO2 nanoparticles.