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Objective: To study the pathogenesis of influenza virus A (H1N1) infected with human immune cells, and quercetin (quercetin) in this study based on antiviral drugs for influenza with oseltamivir (oseltamivir) at different time of influenza virus infection cell proliferation and its mechanism to provide experimental basis for the research and development of new antiviral drugs for influenza. Method: 1. Isolated from fresh normal human umbilical cord blood mononuclear cells, and then induced to differentiate into dendritic cells (DC), macrophages were identified. 2 influenza virus H1N1 A/E13 with chick embryo allantoic cavity inoculation were amplified and measured TCID50; reuse 100TCIDso H1N1 A/E13 infected human bronchial epithelial cells 16HBE cell lines, the establishment of the model of influenza virus-infected cells. MTT assay quercetin oseltamivir Wei in vitro inhibition of influenza virus and its effect relationship (24h, 48h, 72h), to investigate the effect of quercetin and oseltamivir in vitro anti-influenza virus and features determine the optimal concentration of the drug antiviral. Observed quercetin, oseltamivir against influenza virus infection of macrophages release NO level: co-cultured macrophages with influenza virus infection model, and join quercetin, oseltamivir, respectively 6h each group were collected supernatant, 12h, 24h, 48h, nitrate reductase method to detect the level of NO. 5, respectively, DC, macrophages and cell models of influenza virus infection of cultured, quercetin, oseltamivir Wei as therapeutic agents, 24h, 48h collection of immune cells, real-time PCR reaction (RT- qPCR) was measured before and after the viral infection and the use of antiviral drugs DC, TLR7 signal transduction pathway in the macrophage signaling proteins: Toll-like receptor 7 (Toll-like receptor 7, TLR7), myeloid differentiation factor 88 (myeolid differentiationfactor 88 MyD88), interleukin-1 receptor-associated kinase (IL-1 Receptor associated kinase 4, IRAK4), tumor necrosis factor-related kinase 6 (TNF receptor associated kinase TRAF6) and nuclear factor-kappa B (nuclear factor-KB p65, NF-κB p65) mRNA expression. 6, respectively, DC, macrophages and cell models of influenza virus infection of cultured, quercetin, oseltamivir Wei as therapeutic agents, immune cells collected at 24h, Western-bolt method detected before and after the viral infection and the use of DC, macrophages antivirals TLR7, NF-κB p65 protein expression. Results: 1. Successfully isolated from human umbilical cord blood induced dendritic cells, macrophages. Chick embryo allantoic cavity inoculation successfully amplified influenza virus; hemagglutination test measured the virus titer of 1:640; According to the experimental results, according to the Reed-Muench method of influenza virus TCID50 = 5 × 10-4.56. 3 MTT test confirmed quercetin oseltamivir the same anti-influenza virus and measured the optimal drug concentration of both drugs. Influenza virus infection can cause macrophages to release NO levels, quercetin and oseltamivir role of macrophages after influenza virus infection in 6h, 12h, 24h, 48h can be significantly lower activation giant macrophages release NO levels. RT-qPCR experiments prove that influenza virus infection can increase dendritic cells, macrophages TLR7 signaling pathway protein mRNA expression; using quercetin and oseltamivir Wei therapy can significantly lowered the infection of dendritic cells, macrophages, TLR7, MyD88, IRAK4, TRAF6, NF-kappaB mRNA expression levels. Western-blot experiments further evidence of viral infection can be caused by TLR7, NF-kappaB p65 protein expression increased use of quercetin with oseltamivir treatment Wei cut TLR7, NF-kappaB p65 protein expression. Conclusion: influenza virus infection can be caused by dendritic cells and macrophages in vitro TLR7 signal transduction pathway activation, quercetin and oseltamivir can play a role in anti-influenza virus, and reduced activation of TLR7 signaling pathway.
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