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Anti-viral Effects of Silver-nanoparticles on H3N2 Influenza Virus in vitro and Its Mechanism

【摘要】　目的　研究纳米银体外抗H3N2流感病毒的作用，并初步探索其作用机制。　方法　在H3N2流感病毒吸附细胞后加入纳米银和吸附前用纳米银预处理犬肾细胞（MDCK），在体外用细胞病变效应（cytopathic effect，CPE）观察法和3-（4，5-二甲基-2-噻唑）-2，5-二苯基溴化四唑（3-（4，5-dimethylthiazol-2-yl）-2，5-diphenyltetrazolium bromide，MTT）测值法，分析纳米银对H3N2流感病毒感染MDCK细胞的预防作用、直接灭活作用以及对流感病毒子代病毒体生成的抑制作用，运用RT-PCR法研究纳米银对H3N2流感病毒HA基因复制的干扰作用。　结果　纳米银能明显杀伤H3N2流感病毒，50、25 μg/mL的纳米银溶液与H3N2流感病毒充分作用2 h后感染MDCK细胞，细胞存活率分别为94.38%和92.17%，纳米银能有效抑制流感病毒对MDCK细胞的侵入和侵入后病毒的继续增殖，25 μg/mL纳米银溶液通过上述两种方式处理细胞，细胞存活率分别为85.39%和83.28%，与病毒对照组相比，差异均有统计学意义（Plt;0.001）；400、200 μg/mL纳米银溶液分别与流感病毒H3N2充分混合作用15、30、60、120 min后，病毒液的HA基因均未能成功扩增，纯病毒液和溶剂对照组在1 700 bp处均出现明显条带。　结论　通过3种不同的给药方式，纳米银在体外均能明显抑制流感病毒对细胞的感染，纳米银抑制流感病毒的机制可能是通过干扰H3N2流感病毒和吸附、穿入和基因的复制，从而抑制子代病毒体的生成。【Abstract】　Objective　To explore the anti-viral effects of silver-nanoparticles (silver-nps) on H3N2 influenza virus in vitro and to evaluate its mechanism.　Methods　Silver-nps was added to canine kidney cells (MDCK) before and after the cells was adsorpted by H3N2 influenza virus. Cytopathic effect (CPE) assay and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were used to analyze the preventive effect, directly off deactivation, and the inhibit formation of progeny virions of silver-nps on H3N2 viruses. The interference of HA gene replication was observed by the RT-PCR assay.　Results　The survival rate of MDCK cells was 94.38% and 92.17% after 50 and 25 μg/mL silver-nps were mixed with 100 TCID50 H3N2 virus in 2 hours, and the survival rate of MDCK cells was 85.39% and 83.28% before and after the cells was adsorpted by H3N2 influenza virus when 25 μg/mL silver-nps was added to the cells (all compared to virus control, Plt;0.001), which showed that silver-nps could inactivate H3N2 virus, prevente them invasing to the cells and reproducting when H3N2 entered the cell remarkedly. The HA gene was not amplified successfully when 50 and 25 μg/mL silver-nps were mixed with 100TCID50 H3N2 virus in 15, 30, 60, and 120 minutes later, but both pure virus solution and solvent control group appeared a significant bright band in the 1 700 bp area.　Conclusion　Under three different administration modes, silver-nps has an obvious effect against H3N2 in vitro, which could interfere the HA gene replication and inhibit the formation of H3N2 progeny virions.