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目的探索病毒感染产生的中间产物双链RNA(dsRNA)分子刺激对细胞形成应激颗粒(SG)的影响,为进一步研究病毒诱导与拮抗SG形成的分子机制提供实验依据。方法采用不同长度的dsRNA,包括长链(HMW,1.5~8 kb)、短链(LMW,0.2~1 kb)以及小分子dsRNA(19 bp),分别转染A549细胞,模拟病毒感染产生的应激压力。通过免疫荧光检测形成SG的细胞百分率,以及蛋白免疫印迹(WB)检测不同长度的dsRNA诱导抗病毒激酶蛋白激酶R(PKR)及其底物eIF2α的活化水平。结果 HMW、LMW dsRNA可诱导A549细胞产生SG,而小分子dsRNA并不能诱导SG产生。免疫印迹结果发现HMW、LMW dsRNA可诱导PKR和下游底物eIF2α的磷酸化,而小dsRNA不足以活化PKR。结论病毒可能通过降解其dsRNA避免PKR的激活和SG形成,从而逃逸宿主应激应答。
Objective To investigate the effect of dsRNA stimulation on SG cells induced by virus infection and to provide experimental evidence for further study on the molecular mechanism of virus induction and antagonism of SG formation. Methods Different lengths of dsRNA were transfected into A549 cells using long-chain (HMW, 1.5-8 kb), short-chain (LMW, 0.2-1 kb) and small dsRNA (19 bp) Stress. The percentage of SG-forming cells was measured by immunofluorescence and Western blotting (WB) was used to detect dsRNAs of different lengths to induce activation of anti-viral kinase protein kinase R (PKR) and its substrate eIF2α. Results HMW and LMW dsRNA induced A549 cells to produce SG, whereas small molecule dsRNA did not induce SG production. Immunoblotting revealed that HMW and LMW dsRNA induced phosphorylation of PKR and downstream substrate eIF2α, while small dsRNA was insufficient to activate PKR. Conclusion The virus may escape host stress response by degrading its dsRNA to avoid PKR activation and SG formation.