EGCG下调Hsp70抑制呼吸道合胞病毒的活性研究

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目的:探讨表没食子儿茶素没食子酸酯(EGCG)下调热休克蛋白70(Hsp70)对呼吸道合胞病毒(RSV)活性的抑制作用。方法:根据细胞毒性实验确定EGCG的最大工作浓度,采用病毒滴度TCID50检测法、细胞毒性检测法、Western blot和实时荧光定量PCR法(qPCR)检测EGCG体外对RSV的增殖、蛋白表达和基因转录抑制效果。采用RNA干扰敲低Hsp70表达水平,检测病毒的生长曲线。结果:EGCG对Vero细胞的毒性呈梯度依赖效应,最大无毒浓度为25 μm。EGCG在25 μm和12 μm及以上浓度时,呼吸道合胞病毒A型(RSV-A)病毒抑制率达60%以上,EGCG细胞中病毒F蛋白受到显著抑制,EGCG使病毒G蛋白的转录下降了约88%。敲低Hsp70表达水平后,病毒滴度下降了两个数量级。结论:EGCG体外对RSV-A有较好拮抗作用,可能是通过抑制其特异性底物Hsp70实现的。“,”Objective:To study the effect of epigallocatechin-3-gallate (EGCG) on respiratory syncytial virus (RSV) in vitro via down-regulation heat shock protein 70 (Hsp70).Methods:The maximum working concentration of EGCG was determined according to the cytotoxicity experiment. the effects of EGCG on the proliferation, protein expression and gene transcriptional inhibition of RSV in vitro were detected by TCID50 assay, cytotoxicity assay, Western blot, and real-time quantitative PCR (qPCR), respectively. RNA interference was used to knock down the expression level of Hsp70, and the growth curve of the virus was detected.Results:Toxicity of EGCG on Vero cells showed an obvious gradient dependent effect, with the maximum non-toxic concentration of 25 μm. When EGCG was at 25 μm and 12 μm and above, the respiratory syncytial virus type A (RSV-A) virus inhibition rate reached more than 60%. The virus F protein in EGCG treated cells was significantly inhibited, EGCG reduced the transcription efficiency of virus G protein by about 88%. Knocking down the expression level of Hsp70 reduced the titer of the virus by two orders of magnitude.Conclusions:EGCG has a good antiviral effect on RSV-A in vitro, and may be achieved by inhibiting its specific substrate Hsp70.
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