纳米银抗流感病毒（H3N2）作用及机制研究

发布时间：2018-01-16 10:41

本文关键词：纳米银抗流感病毒（H3N2）作用及机制研究　出处：《大连大学》2011年硕士论文　论文类型：学位论文

【摘要】：流感病毒(Influenza virus, IFV)属正粘科属病毒,流感病毒的感染能够引起严重的呼吸道疾病,对人类健康的威胁不仅表现为造成个体严重感染甚至死亡,更因为流感病毒可以经过频繁变异,突变成高致病毒株或获得动物和人之间、人和人之间快速传播的能力。因此,需要研发有效的药物应对流感病毒的潜在威胁。纳米银(Silver nanoparticles,以下简称Silver-nps)可以抑制人类免疫缺陷性病毒和乙型肝炎病毒的活性。Silver-nps对流感病毒的抑制作用及其机制如何,尚缺乏研究。本文旨在研究Silver-nps对H3N2亚型流感病毒(以下简称H3N2-IFV)是否具有抑制作用,并探讨其作用机制。在体外将H3N2-IFV和Silver-nps相互作用后,直接检测H3N2-IFV的血凝素效价,结果显示,对照组病毒的血凝效价为1:1024,H3N2-IFV与Silver-nps相互作用30min,60min,90min和120min后的血凝效价分别为1:16,1:8,1:4和低于1:2;通过鸡胚培养H3N2-IFV,经Silver-nps处理后的H3N2-IFV血凝效价明显低于对照组(below 1:2 vs. 1:1024, P 0.001),结果提示Silver-nps可能破坏H3N2-IFV表面的血凝素蛋白。通过神经氨酸酶活性抑制试验,Silver-nps能够抑制神经氨酸酶活性物质的释放,表明Silver-nps可能破坏了流感病毒表面的神经氨酸酶蛋白。运用细胞培养技术,通过CPE观察法、MTT测值法和免疫荧光观察法,分析Silver-nps对H3N2-IFV感染犬肾细胞(MDCK)细胞的预防作用、直接灭活作用以及对H3N2-IFV子代病毒体生成的抑制作用(治疗作用),结果显示,与对照组细胞内强特异性荧光相比,在上述作用的三种途径中细胞内特异性荧光均很少见,Silver-nps能够明显灭活H3N2-IFV,并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。通过透射电镜负染技术观察到Silver-nps可以直接破坏H3N2-IFV颗粒的包膜,说明Silver-nps颗粒可能通过直接破坏流感病毒的形态和结构来影响病毒的功能。同时,通过透射电镜和流式细胞术发现,Silver-nps能抑制H3N2-IFV诱导MDCK细胞发生凋亡的作用,结果显示,Silver-nps作用病毒组中的细胞形态结构基本正常,病毒对照组细胞可见明显凋亡现象,三种途径处理组中细胞凋亡率均明显低于对照组,差异具有统计学意义(P 0.05)。最后通过RT-PCR法,结果显示,Silver-nps能够干扰H3N2-IFV HA基因的扩增,而对照组均可见明显的条带,表明Silver-nps可能干扰了病毒遗传物质复制的某个环节。上述实验结果表明,Silver-nps在细胞水平和分子水平上对流感病毒均具有抑制作用,本研究可为成功研制Silver-nps抗病毒药物和用于临床防治流感提供实验基础和理论依据。
[Abstract]:Influenza virus Influenza virus (IFV) is an orthomyxidae virus. Influenza virus infection can cause serious respiratory diseases. The threat to human health is not only to cause serious individual infection or even death, but also because influenza viruses can mutate frequently, mutate into highly pathogenic strains or get between animals and people. The ability to spread rapidly from person to person. Therefore, it is necessary to develop effective drugs to deal with the potential threat of influenza virus. Silver nanoparticles. Silver-nps. can inhibit the activity of human immunodeficiency virus and hepatitis B. Silver-nps inhibits influenza virus and its mechanism. The purpose of this study is to investigate whether Silver-nps has inhibitory effect on H3N2 subtype influenza virus (H3N2-IFV). After the interaction between H _ 3N _ 2-IFV and Silver-nps in vitro, the hemagglutinin titer of H _ 3N _ 2-IFV was detected directly. The hemagglutination titer of the control group was 1: 1024 H3N2-IFV interacting with Silver-nps for 30 min or 60 min. After 90 min and 120 min, the titer of hemagglutination was 1: 16: 1: 8: 1: 4 and less than 1: 2, respectively. H3N2-IFV was cultured from chicken embryo. The hemagglutination titer of H3N2-IFV treated with Silver-nps was significantly lower than that of control group (1: 2 vs 1: 1024, P 0.001). The results suggest that Silver-nps may destroy the hemagglutinin protein on the surface of H3N2-IFV and inhibit the activity of neuraminidase. Silver-nps can inhibit the release of neuraminidase activity, suggesting that Silver-nps may destroy the neuraminidase protein on the surface of influenza virus. The preventive effect of Silver-nps on H3N2-IFV infected canine renal cell line (MDCK) was analyzed by CPE assay and immunofluorescence assay. Direct inactivation and inhibition of virus formation in H3N2-IFV progeny (therapeutic effect), the results showed that compared with the control group, the intracellular strong specific fluorescence was higher than that of the control group. In these three pathways, intracellular specific fluorescence was rare. Silver-nps could significantly inactivate H3N2-IFV. It can effectively inhibit the invasion of influenza virus to MDCK cells and the continuous proliferation of virus after invasion. It was observed by transmission electron microscopy that Silver-nps can directly destroy H3N2-IFV particles. The capsule of a particle. It is suggested that Silver-nps particles may affect the function of influenza virus by directly destroying the morphology and structure of influenza virus. At the same time, transmission electron microscopy and flow cytometry were used to detect the function of the virus. Silver-nps could inhibit the apoptosis of MDCK cells induced by H3N2-IFV. The results showed that the morphology of MDCK cells in Silver-nps treated group was basically normal. The apoptosis rate of the virus control group was significantly lower than that of the control group, and the difference was statistically significant (P 0.05). Finally, the RT-PCR method was used. The results showed that Silver-nps could interfere with the amplification of H _ 3N _ 2-IFV HA gene, but there were obvious bands in the control group. These results suggest that Silver-nps may interfere with some part of viral genetic material replication. Silver-nps can inhibit influenza virus at both cellular and molecular level. This study can provide experimental and theoretical basis for the successful development of Silver-nps antiviral drugs and clinical prevention and treatment of influenza.
【学位授予单位】：大连大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373

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