F81细胞外囊泡介导IFN-ω上调抗病毒细胞因子表达的研究

F81 extracellular vesicles mediate upregulation of antiviral cytokine expression by IFN-ω

对猫肾细胞(F81)外囊泡(extracellular vesicles,EVs)介导猫omega干扰素(interferon omega,IFN-ω)调控抗病毒细胞因子变化的功能研究,为EVs介导的干扰素抗病毒机制研究提供理论基础。提取IFN-ω刺激F81细胞分泌的IFN-ω-EVs,通过Western-blotting、透射电镜、纳米粒径技术分别对IFN-ω-EVs的标志蛋白、形态、粒径进行鉴定;将携带IFN-ω的IFN-ω-EVs孵育到F81细胞,以激光共聚焦显微镜进行荧光共定位观察分析IFN-ω-EVs是否进入F81细胞,通过RT-q PCR检测ISG15、ISG56、IFN-ω细胞因子的表达水平,与IFN-ω、空白对照进行对比,以此研究IFN-ω-EVs是否对水疱性口炎病毒(VSV)具有抗病毒效应。经鉴定IFN-ω-EVs主要为杯状囊结构,携带特异性标记蛋白CD63、CD81,其粒径在50~100、250~600 nm之间;荧光共定位分析发现IFN-ω-EVs能够进入F81细胞;RT-q PCR结果表明,IFN-ω-EVs同IFN-ω一样能显著上调ISG15、ISG56、IFN-ω等抗病毒细胞因子的表达水平。重要的是VSV感染后,IFN-ω-EVs的抗VSV作用显著高于IFN-ω(P<0.05)。上述结果表明,F81提取得到的IFN-ω-EVs能够介导IFN-ω进入F81细胞中,并显著上调ISG15、ISG56、IFN-ω等抗病毒细胞因子的表达,抑制VSV增殖。

This study aims to investigate the role of extracellular vesicles(EVs)from feline kidney cells(F81)in mediating the regulation of antiviral cytokines through feline omega interferon(IFN-ω),providing a theoretical foundation for research on EV-mediated interferon antiviral mechanisms.IFN-ω-stimulated IFN-ω-EVs secreted by F81 cells were extracted and characterized for their marker proteins,morphology,and size using Western blotting,transmission electron microscopy,and nanoscale particle technology,respectively.F81 cells were incubated with IFN-ω-EVs containing IFN-ω,and fluorescence colocalization was analyzed using a laser confocal microscope to determine whether IFN-ω-EVs enter F81 cells.The expression levels of ISG15,ISG56,and IFN-ω cytokines were detected by RT-q PCR to investigate the antiviral effects of IFN-ω-EVs against VSV,in comparison with IFN-ω and a blank control.IFN-ω-EVs were primarily identified as cup-shaped vesicular structures carrying specific marker proteins CD63 and CD81,with particle sizes ranging between 50—100 nm and 250—600 nm.Fluorescence colocalization analysis revealed that IFN-ω-EVs could enter F81 cells.RT-q PCR results indicated that IFN-ω-EVs,like IFN-ω,significantly upregulated the expression levels of antiviral cytokines such as ISG15,ISG56,and IFN-ω.Importantly,the anti-VSV activity of IFN-ω-EVs post-VSV infection was significantly higher than that of IFN-ω alone(P<0.05).IFN-ω-EVs extracted from F81cells,can mediate the entry of IFN-ω by F81 cells,significantly upregulate the expression of antiviral cytokines such as ISG15,ISG56,and IFN-ω,and inhibit the inhibition of VSV virus proli-feration.