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为了揭示肠道病毒71型(enterovirus71,EV71)的复制与宿主细胞Raf/MEK/ERK信号通路(简称ERK通路)的相互关系,本研究应用临床诊断为手足口病的患儿疱疹液,通过易感细胞分离培养、RT-PCR及序列测定,以及Western印迹技术等方法,成功分离到EV71临床株.进一步用该分离株感染易感细胞,通过观察宿主细胞p-ERK1/2蛋白磷酸化水平、病毒特异性衣壳蛋白VP1水平、病毒半数组织培养感染量(50%tissue culture infectious dose,TCID50),以及感染细胞的CPE等指标,以期揭示ERK通路在EV71复制的作用.结果表明,EV71的复制可引起细胞ERK通路的活化;而用MEK1/2特异性的抑制剂U0126预先抑制ERK通路的活化,可显著地降低受染细胞上清液中的病毒的感染滴度(以TCID50表示)、受染细胞中EV71VP1蛋白水平、受染细胞中EV71核酸水平,以及受染细胞的细胞病变效应(cytopathic effect,CPE).提示ERK信号通路的活化对EV71的复制具有重要的作用.本研究为进一步阐明EV71在宿主细胞内的复制机制、寻找新型抗病毒靶标等研究奠定了良好的基础.
In order to reveal the relationship between the replication of enterovirus 71 (EV71) and the Raf / MEK / ERK signaling pathway in host cells (ERK pathway), this study used herpes simplex in patients with HFMD clinically. The clinical isolates of EV71 were successfully isolated from the infected cells by RT-PCR, sequence analysis and Western blotting.The susceptible cells were further infected by this isolate, and the phosphorylation of p-ERK1 / 2 in host cells was observed, VP1 level of virus-specific capsid protein, 50% tissue culture infectious virus (TCID50) and CPE of infected cells in order to reveal the role of ERK pathway in EV71 replication.The results showed that EV71 replication Can induce the activation of ERK pathway in cells; while pretreatment of ERK pathway with MEK1 / 2-specific inhibitor U0126 can significantly reduce the virus titer (expressed as TCID50) in infected cell supernatant by EV71VP1 protein levels in infected cells, levels of EV71 nucleic acid in infected cells, and cytopathic effect (CPE) of infected cells suggest that activation of the ERK signaling pathway has the ability to replicate EV71 Important role.This study laid a good foundation for further elucidating the mechanism of EV71 replication in host cells and searching for new antiviral targets.