王秀娟^1,2,3,
李婷竹^1,2,3,
陆强^1,2,3,
苏雪荣^1,2,3,
薛玉英^1,2,3,
唐萌^1,2,3
1. 环境医学工程教育部重点实验室, 南京 210009;
2. 东南大学公共卫生学院 & 苏州纳米科技协同创新中心, 南京 210009;
3. 江苏省生物材料与器件重点实验室, 南京 210009

作者简介: 王秀娟(1993-),女,硕士研究生,研究方向为纳米毒理学,E-mail:924153949@qq.com.

基金项目: 国家自然科学基金项目(81573186,81473003)
大学生创新创业计划训练项目(201710286130)


中图分类号: X171.5

Comparative Study on the Genetic Toxicity of Nano Silver and PVP Coated Silver Nanoparticles to HepG2 Cells

Wang Xiujuan^1,2,3,
Li Tingzhu^1,2,3,
Lu Qiang^1,2,3,
Su Xuerong^1,2,3,
Xue Yuying^1,2,3,
Tang Meng^1,2,3
1. Key Laboratory of Environmental Medicine and Engineering of Ministry of Education, Nanjing 210009, China;
2. School of Public Health of Southeast University & Collaborative Innovation Center of Suzhou Nanoscience and Technology, Nanjing 210009, China;
3. Jiangsu Key Laboratory for Biomaterials and Devices, Nanjing 210009, China

CLC number: X171.5

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摘要:为了探讨纳米银对HepG2细胞DNA损伤、染色体畸变等遗传毒性指标的影响，以期为纳米银体外遗传毒性评价提供参考依据，本文采用2种纳米银材料(20 nm-PVP包被纳米银、20 nm-无包被纳米银)，分别以20 μg·mL^-1、40 μg·mL^-1、80 μg·mL^-1、160 μg·mL^-1的剂量对HepG2细胞染毒24 h，用Hoechst-33258染色法检测细胞凋亡，彗星实验检测DNA损伤，胞质分裂阻滞微核细胞组学试验法检测染色体畸变。结果表明，20 nm AgNPs组在160 μg·mL^-1时引起细胞凋亡数显著增多(P<0.05)；20 nm PVP-AgNPs组在80 μg·mL^-1和160 μg·mL^-1剂量组中细胞凋亡数显著增多(P<0.01)。2种纳米银引起HepG2细胞发生细胞凋亡，并呈剂量效应关系。彗星试验结果表明，20 nm AgNPs和20 nm PVP-AgNPs在40 μg·mL^-1、80 μg·mL^-1、160 μg·mL^-1剂量组中，Olive尾矩、尾长和尾部DNA百分比与空白对照组相比均有显著差异(P<0.05)。2种纳米银对HepG2细胞DNA损伤程度为：20 nm AgNPs>20 nm PVP-AgNPs。胞质分裂阻滞微核细胞组学试验结果表明，2种纳米银均不会引起核质桥数发生明显改变(P>0.05)，20 nm AgNPs在高染毒剂量下引起微核总数、I型微核、II型微核、核芽数明显升高(P<0.05)；20 nm PVP-AgNPs在各染毒剂量下均会引起微核总数及I型微核数量升高(P<0.01)，II型微核数在160 μg·mL^-1剂量下升高明显(P<0.01)，剂量大于20 μg·mL^-1 时核芽数升高(P<0.01)。20 nm PVP-AgNPs对细胞核的影响大于20 nm AgNPs(P<0.05)。总之，2种纳米银材料均会引起HepG2细胞DNA损伤及染色体畸变等遗传毒性效应的改变，无包被纳米银比PVP包被纳米银更容易引起DNA损伤，PVP包被纳米银比无包被纳米银更容易引起细胞染色体畸变相关效应；2种材料对HepG2细胞的损伤存在浓度-效应关系，浓度越高遗传毒性损伤越严重。
关键词: 纳米银/
HepG2细胞/
彗星实验/
微核/
剂量-效应关系

Abstract:The aim of this study was to explore the genetic toxicity of nano silver, such as DNA damage and chromosomal aberration, in HepG2 cells and provide a reference for the in vitro evaluation of genetic toxicity. The HepG2 cells were infected with nano silver (20 nm AgNPs and 20 nm PVP-AgNPs) with a series of doses of 20 μg·mL^-1, 40 μg·mL^-1, 80 μg·mL^-1 and 160 μg·mL^-1 for 24 hours. The cell apoptosis was detected by the hoechst-33258 staining, the DNA damage was detected by the comet assay, and the chromosome aberration was detected by the cytoplasmic block micronuclear assay. Results showed that the number of apoptosis was significantly increased in the 160 μg·mL^-1 20 nm AgNPs group compared with the control (P<0.05), while the numbers of apoptotic cells in the 80 μg·mL^-1 and 160 μg·mL^-1 20 nm PVP-AgNPs group were significantly increased compared to the control (P<0.05, P<0.01). Two kinds of silver nanoparticles induced apoptosis in HepG2 cells in a concentration dependent manner. In the comet test, olive tail moment, tail length and tail DNA percentage in the 40 μg·mL^-1, 80 μg·mL^-1 and 160 μg·mL^-1 20 nm AgNPs and 20 nm PVP-AgNPs were all significantly different from those in the blank control group (P<0.05). The DNA damage degree of HepG2 cells in 20 nm AgNPs was higher than that in 20 nm PVP-AgNPs. In the cytoplasmic block micronucleus assay, neither of silver nanoparticles caused any significant change in the number of cytoplasmic bridge (P>0.05). However, the number of micronucleus, type I micronucleus, type II micronucleus and nucleation buds were significantly increased when cells were exposed to 20 nm AgNPs with the high exposure dose (P<0.05). For the PVP-AgNPs exposure, the total number of micronucleus and type I micronucleus count were all increased when cells were exposed to 20 nm PVP-AgNPs with the three doses (P<0.01). The type II micronucleus count increased significantly in 160 μg·mL^-1 20 nm PVP-AgNPs group (P<0.01), and the nuclear buds number increased obviously in cells treated with PVP-AgNPs at more than 20 μg·mL^-1 (P<0.01). Generally, the toxic effects of 20 nm PVP-AgNPs on the nucleus were severer than 20 nm AgNPs (P<0.05). We concluded that the two types of silver nanoparticles could cause genetic toxicity to HepG2 cells, such as DNA damage and chromosomal aberration. The ability of 20 nm AgNPs causing DNA damage was stronger than 20 nm PVP-AgNPs, while the ability of 20 nm PVP-AgNPs causing chromosomal aberration was stronger than 20 nm AgNPs. Overall, the toxic effects induced by both of AgNPs was in a dose dependent manner, which means higher concentration of nanoparticles induces severer genotoxicity damage in HepG2 cells.
Key words:nano silver/
comet assay/
micronucleus/
dose-response relationship/