抗病毒sirna对体外细胞因子产生的影响

A. V. Pak, E. Pashkov, N. Abramova, A. Poddubikov, F. G. Nagieva, E. Bogdanova, E. P. Pashkov, O. Svitich, V. Zverev
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The nonparametric Mann–Whitney test was used to statistically calculate significant differences between groups.Results. The use of each small interfering ribonucleic acid (siRNA) complex led to a decrease in viral reproduction on the first day at the multiplicity of infection (MOI) of 0.001. The use of complex A (FLT4.2 + Nup98.1) and D (FLT4.2 + Nup98.1 + Nup205) led to a decrease in viral titer by 2.8 lgTCID50/mL and by 2.1 lgTCID50/mL relative to the use of nonspecific L2 siRNA and viral control (p ≤ 0.05). Transfection of complexes B (Nup98.1 + Nup205) and C (FLT4.2 + Nup205) also reduced the viral titer by 1.5 lgTCID50/mL and 1.8 lgTCID50/mL relative to nonspecific L2 siRNA and viral control (p ≤ 0.05). When conducting real-time RT-qPCR, a significant decrease in the concentration of viral RNA was also noted. When using complexes B, C, and D, the concentration of vRNA decreased on the first day by 14.5, 4.1, and 15 times, respectively. On the second day, a decrease in vRNA was observed in cells with B and D complexes by 17.1 and 18.3 times (p ≤ 0.05). Along with a decrease in the viral titer and vRNA, an increase in the expression of the IL-1β and IL-28β genes was observed on the first day when using all siRNA complexes relative to nonspecific and viral controls (p ≤ 0.05). On the second day, an increase was also observed in cells with A and D complexes, while on the third day, there was an increase in the expression of these genes in cells with complex D (p ≤ 0.05).Conclusions. The use of siRNA complexes is shown to have a pronounced antiviral effect while simultaneously suppressing the activity of cellular genes (FLT4, Nup98 and Nup205). In parallel, the transfection of complexes that block the formation of expression products necessary for viral reproduction is demonstrated to lead to an increase in the level of expression of the IL-1β and IL-28β genes. 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引用次数: 1

摘要

目标。目的探讨IL-1β和IL-28β (IFN-λ3)基因在流感病毒复制过程中表达的动态变化。在转染和感染后3天内收集含病毒液和细胞裂解液,使用细胞病变效应滴定法评估病毒繁殖强度。实时逆转录定量聚合酶链反应(real-time RT-qPCR)检测病毒核糖核酸(vRNA)浓度及IL-1β和IL-28β (IFN-λ3)表达变化。采用非参数Mann-Whitney检验统计各组间的显著差异。每个小干扰核糖核酸(siRNA)复合物的使用导致病毒繁殖在第一天减少,感染多重性(MOI)为0.001。与使用非特异性L2 siRNA和病毒对照相比,使用复合物A (FLT4.2 + Nup98.1)和D (FLT4.2 + Nup98.1 + Nup205)导致病毒滴度分别降低2.8 lgTCID50/mL和2.1 lgTCID50/mL (p≤0.05)。与非特异性L2 siRNA和病毒对照相比,转染复合物B (Nup98.1 + Nup205)和C (FLT4.2 + Nup205)也使病毒滴度降低了1.5 lgTCID50/mL和1.8 lgTCID50/mL (p≤0.05)。在进行实时RT-qPCR时,还注意到病毒RNA浓度显著降低。当使用配合物B、C和D时,vRNA浓度在第一天分别下降14.5倍、4.1倍和15倍。第2天,含有B和D复合物的细胞vRNA分别减少17.1倍和18.3倍(p≤0.05)。随着病毒滴度和vRNA的降低,与非特异性和病毒对照相比,使用所有siRNA复合物的第一天IL-1β和IL-28β基因的表达增加(p≤0.05)。第2天,A、D复合物细胞表达量增加,第3天,A、D复合物细胞表达量增加(p≤0.05)。使用siRNA复合物被证明具有明显的抗病毒作用,同时抑制细胞基因(FLT4, Nup98和Nup205)的活性。与此同时,转染阻断病毒繁殖所需表达产物形成的复合物可导致IL-1β和IL-28β基因表达水平的增加。这些结果表明,siRNA的使用不仅具有抗病毒活性,而且具有免疫调节活性,可以促进机体更有效的免疫反应。
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Effect of antiviral siRNAs on the production of cytokines in vitro
Objectives. To evaluate the dynamics of the expression level of IL-1β and IL-28β (IFN-λ3) genes as a result of complex knockdown of some cellular genes, whose expression products play an important role in the reproduction of the influenza virus.Methods. Following the collection of virus-containing liquid and cell lysate within three days from the moment of transfection and infection, the intensity of viral reproduction was assessed using the cytopathic effect titration method. The concentration of viral ribonucleic acid (vRNA) and change in the expression of IL-1β and IL-28β (IFN-λ3) were determined by real-time reverse transcription quantitative polymerase chain reaction (real-time RT-qPCR). The nonparametric Mann–Whitney test was used to statistically calculate significant differences between groups.Results. The use of each small interfering ribonucleic acid (siRNA) complex led to a decrease in viral reproduction on the first day at the multiplicity of infection (MOI) of 0.001. The use of complex A (FLT4.2 + Nup98.1) and D (FLT4.2 + Nup98.1 + Nup205) led to a decrease in viral titer by 2.8 lgTCID50/mL and by 2.1 lgTCID50/mL relative to the use of nonspecific L2 siRNA and viral control (p ≤ 0.05). Transfection of complexes B (Nup98.1 + Nup205) and C (FLT4.2 + Nup205) also reduced the viral titer by 1.5 lgTCID50/mL and 1.8 lgTCID50/mL relative to nonspecific L2 siRNA and viral control (p ≤ 0.05). When conducting real-time RT-qPCR, a significant decrease in the concentration of viral RNA was also noted. When using complexes B, C, and D, the concentration of vRNA decreased on the first day by 14.5, 4.1, and 15 times, respectively. On the second day, a decrease in vRNA was observed in cells with B and D complexes by 17.1 and 18.3 times (p ≤ 0.05). Along with a decrease in the viral titer and vRNA, an increase in the expression of the IL-1β and IL-28β genes was observed on the first day when using all siRNA complexes relative to nonspecific and viral controls (p ≤ 0.05). On the second day, an increase was also observed in cells with A and D complexes, while on the third day, there was an increase in the expression of these genes in cells with complex D (p ≤ 0.05).Conclusions. The use of siRNA complexes is shown to have a pronounced antiviral effect while simultaneously suppressing the activity of cellular genes (FLT4, Nup98 and Nup205). In parallel, the transfection of complexes that block the formation of expression products necessary for viral reproduction is demonstrated to lead to an increase in the level of expression of the IL-1β and IL-28β genes. These results indicate not only that the use of siRNA has antiviral activity, but also immunomodulatory activity, which can contribute to a more effective immune response of the body.
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