外源性外泌体对mTOR通路参与柯萨奇病毒B3诱导细胞凋亡的调控作用及机制研究

项目名称： 外源性外泌体对mTOR通路参与柯萨奇病毒B3诱导细胞凋亡的调控作用及机制研究

项目编号： No.81500225

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 医药、卫生

项目作者： 李欣

作者单位： 中南大学

项目金额： 18万元

中文摘要： 研究表明细胞外外泌体（Exos）在细胞增殖与凋亡、病毒感染性疾病等生物进程中发挥作用。但是Exos是否可调节病毒感染后诱导产生的细胞凋亡目前尚未见报道。课题组前期研究表明，mTOR通路参与CVB3诱导的细胞凋亡，且国内外研究显示Exos可以调控病毒复制及进出细胞并能作用于Akt通路而调控细胞凋亡等生命进程。因此我们推测Exos可能通过磷酸化mTOR通路、抑制病毒复制，从而减轻CVB3诱导的细胞凋亡。为证实此项假设，本研究拟构建mTOR通路抑制及过表达细胞模型，同时使用Exos及CVB3干预，通过检测凋亡因子、mTOR通路信号分子、病毒复制及释放以及Exos中miRNA变化等，观察病毒感染后不同时间点细胞凋亡、mTOR通路及病毒复制的变化，以初步阐释Exos在该通路参与CVB3诱导的细胞凋亡中的作用，为进一步研究CVB3诱导细胞凋亡的作用机制提供新的理论依据，同时为其治疗提供新的思路及靶点。

中文关键词： 细胞外泌体；mTOR信号通路；柯萨奇病毒B3；细胞凋亡

英文摘要： Recent evidences corroborate that extracellular exosomes play a role in several biological processes, including inhibiting the cell apoptosis, activating PI3K/Akt pathway to enhance the cellular viability, participating in the viral pathogenesis, and so on. However, whether extracellular exosomes involve the regulation of the virus induced apoptosis is still an enigma. Given that our previous studies had performed that PI3K/Akt/mTOR signaling pathway participated in the Coxsackie virusB3 (CVB3) induced apoptosis, it is conceivable to consider that extracellular exosomes may take part in the CVB3 induced apoptosis via regulation of the mTOR signaling pathway together with the virus replication. To illustrate the hypothesis, in this study, extracellular exosomes will be added in the cells with or without the inhibitors of PI3K and mTOR or the overexpression of 4EBP1, p70S6K, Akt1 or Akt2 before CVB3 infection. Then, the change of the cytopathic effect and apoptosis in different infected time will be detected by transmission electron microscope. Furthermore, the change of the apoptosis and the PI3K/Akt/mTOR pathway will be investigated by the Real-time fluorescence quantitative PCR and Western blot analysis, respectively. Finally, we will examine the replication and release of CVB3, together with the change of the miRNA in exosomes at 24 h postinfected(p. i.). Our study will originally and preliminarily elucidate thepossible effects of extracellular exosomes in the CVB3 induced cell apoptosis, which may provide a new perspective of the mechanism and the development of new therapy of CVB3-induced apoptosis.

英文关键词： exosomes;mTOR signaling pathway;CVB3;apoptosis