纳米二氧化硅颗粒对肝HL-7702细胞的毒性作用

Cytotoxicity of SiO_2 nanoparticles on liver HL-7702 cells

目的:探讨纳米二氧化硅(SiO2)颗粒对肝细胞HL-7702的毒性作用,阐明其作用机制。方法:体外培养的人正常HL-7702肝细胞随机分为阴性对照组和12.5、25.0、50.0、100.0mg·L-1 SiO2组。采用透射电子显微镜(TEM)和动态光散射(DLS)法对纳米SiO2颗粒进行表征和粒径检测。细胞处理24h后,HE染色观察各组细胞形态学;MTT实验检测各组细胞存活率;乳酸脱氢酶(LDH)活力实验检测各组细胞培养液中LDH活力;流式细胞术(FCM)检测各组细胞中活性氧(ROS)水平。结果:TEM观察,纳米SiO2呈球形,颗粒大小较均匀一致,分散性较好,颗粒平均粒径为(65.87±9.02)nm;DLS法检测,纳米SiO2颗粒在高纯水和RPMI-1640培养液中的水合粒径明显增大,分别为(124.57±8.02)和(139.32±9.93)nm。HE染色,纳米SiO2颗粒能够导致HL-7702细胞形态改变,25.0和50.0 mg·L-1 SiO2组细胞数目减少,排列紊乱;100.0mg·L-1 SiO2组细胞质皱缩、细胞排列稀疏,部分细胞呈凋亡的形态学表现。与阴性对照组比较,各浓度纳米SiO2组HL-7702细胞存活率均下降,50.0和100.00mg·L-1 SiO2组细胞存活率明显下降(P<0.05);50.0和100.00mg·L-1 SiO2组培养液中LDH活力明显升高(P<0.05),细胞中ROS水平明显升高(P<0.05)。结论:纳米SiO2颗粒能够对HL-7702细胞产生毒性作用,并呈一定的浓度依赖效应;其毒性作用机制可能与细胞中ROS的产生有关联。

Objective To study the cytotoxicity of SiO2 nanoparticles （NPs） on the liver HL-7702 cells, and to clarify its mechanism. Methods The in vitro cultured human normal liver HL-7702 cells were randomly divided into control group and 12.5, 25.0, 50. 0, 100. 0 mg·L^-1 SiO2 groups, respectively. Transmission electron microscope （TEM） was used to characterize the SiOz NPs, and dynamic light scattering （DLS） method was used to analyze the particle size of SiO2 NPs. After 24 h exposure, the morphology of HL-7702 cells were observed by HE staining; MTT assay was used to determine the viability of HL-7702 cells; lactate dehydrogenase （LDH） release assay was used to detect the LDH activity in culture medium of HL-7702 cells; flow cytometry （FCM） was applied to examine the level of intraeellular reactive oxygen species （ROS）. Results The TEM results showed that the SiO2 NPs were spherical, universally sized and more or less dispersed well, and the average particle size of SiO2 NPs were （65.87±9.02） nm. The DLS method results manifested that the SiO2 particle sizes of the SiO2 NPs were （124.57±8. 02） nm in high purity water and （139.32±9.93） nm in RPMI-1640 solution. The HE staining results revealed that the SiO2 NPs could cause the morphological changes of HL-7702 cells, and the number of HL-7702 cells was reduced and the arrangement was disorder in 25.0 and 50.0 mg ·L^-1 SiO2 groups; in 100.0 mg·L^-1 SiO2 group, the cells were shrunken and sparse, and part of the cells appeared the morphological features of apoptosis. Compared with control group, the cell viabilities in different concentrations of SiO2 groups were decreased, especialy in 50.0 and 100.0 mg ·L^-1 SiO2 groups （P〈0.05）, while the LDH activities in culture medium and the intracellular ROS levels in 50.0 and 100.0 mg·L^-1 SiO2 groups were increased （P〈0.05）. Conclusion SiO2 NPs could cause the cytotoxieity on the HL-7702 cells in a concentration-dependent manner, and the eytotoxicity may be associated with the generation of intracellular ROS.