本文關鍵詞：納米銀抗流感病毒（H3N2）作用及機制研究　出處：《大連大學》2011年碩士論文　論文類型：學位論文

  更多相關文章： 納米銀 流感病毒 H3N2亞型 抗病毒 機制研究

【摘要】：流感病毒(Influenza virus, IFV)屬正粘科屬病毒,流感病毒的感染能夠引起嚴重的呼吸道疾病,對人類健康的威脅不僅表現(xiàn)為造成個體嚴重感染甚至死亡,更因為流感病毒可以經(jīng)過頻繁變異,突變成高致病毒株或獲得動物和人之間、人和人之間快速傳播的能力。因此,需要研發(fā)有效的藥物應對流感病毒的潛在威脅。納米銀(Silver nanoparticles,以下簡稱Silver-nps)可以抑制人類免疫缺陷性病毒和乙型肝炎病毒的活性。Silver-nps對流感病毒的抑制作用及其機制如何,尚缺乏研究。本文旨在研究Silver-nps對H3N2亞型流感病毒(以下簡稱H3N2-IFV)是否具有抑制作用,并探討其作用機制。在體外將H3N2-IFV和Silver-nps相互作用后,直接檢測H3N2-IFV的血凝素效價,結果顯示,對照組病毒的血凝效價為1:1024,H3N2-IFV與Silver-nps相互作用30min,60min,90min和120min后的血凝效價分別為1:16,1:8,1:4和低于1:2;通過雞胚培養(yǎng)H3N2-IFV,經(jīng)Silver-nps處理后的H3N2-IFV血凝效價明顯低于對照組(below 1:2 vs. 1:1024, P 0.001),結果提示Silver-nps可能破壞H3N2-IFV表面的血凝素蛋白。通過神經(jīng)氨酸酶活性抑制試驗,Silver-nps能夠抑制神經(jīng)氨酸酶活性物質的釋放,表明Silver-nps可能破壞了流感病毒表面的神經(jīng)氨酸酶蛋白。運用細胞培養(yǎng)技術,通過CPE觀察法、MTT測值法和免疫熒光觀察法,分析Silver-nps對H3N2-IFV感染犬腎細胞(MDCK)細胞的預防作用、直接滅活作用以及對H3N2-IFV子代病毒體生成的抑制作用(治療作用),結果顯示,與對照組細胞內強特異性熒光相比,在上述作用的三種途徑中細胞內特異性熒光均很少見,Silver-nps能夠明顯滅活H3N2-IFV,并能有效抑制流感病毒對MDCK細胞的侵入和侵入后病毒的繼續(xù)增殖。通過透射電鏡負染技術觀察到Silver-nps可以直接破壞H3N2-IFV顆粒的包膜,說明Silver-nps顆�？赡芡ㄟ^直接破壞流感病毒的形態(tài)和結構來影響病毒的功能。同時,通過透射電鏡和流式細胞術發(fā)現(xiàn),Silver-nps能抑制H3N2-IFV誘導MDCK細胞發(fā)生凋亡的作用,結果顯示,Silver-nps作用病毒組中的細胞形態(tài)結構基本正常,病毒對照組細胞可見明顯凋亡現(xiàn)象,三種途徑處理組中細胞凋亡率均明顯低于對照組,差異具有統(tǒng)計學意義(P 0.05)。最后通過RT-PCR法,結果顯示,Silver-nps能夠干擾H3N2-IFV HA基因的擴增,而對照組均可見明顯的條帶,表明Silver-nps可能干擾了病毒遺傳物質復制的某個環(huán)節(jié)。上述實驗結果表明,Silver-nps在細胞水平和分子水平上對流感病毒均具有抑制作用,本研究可為成功研制Silver-nps抗病毒藥物和用于臨床防治流感提供實驗基礎和理論依據(jù)。
[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.
【學位授予單位】：大連大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R373

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