细胞自噬在DENV-2感染HUVEC复制中的作用及其AMPK/ERK/mTOR信号通路的研究

前期研究证明，DENV2可以诱导传代HUVEC发生自噬，在胞浆内DENV2 NS1与自噬标记性蛋白LC3B、溶酶体标记蛋白LAMP1共定位，但其信号通路机制不明。本研究拟利用分子生物学，蛋白组学、基因敲除等技术，通过①观察DENV感染HUVEC不同时期对宿主细胞自噬的影响；②使用自噬及其信号通路抑制剂、促进剂，检测自噬对病毒复制、释放及感染的动力学和对宿主增殖和功能的影响；③通过对DENV2感染HUVEC诱导自噬AMPK/ERK/mTOR信号通路进行深入研究，寻找DENV2诱导HUVEC凋亡的靶位点。④构建靶位点基因敲除HUVEC细胞系和小鼠，通过体内外实验，验证DENV-2感染后导致HUVEC功能障碍、病毒增殖与自噬的相关性,靶位点基因敲除小鼠免疫应答类型的特点?旨在为DENV感染发病机制的阐明和治疗提供充分的科学依据，明确自噬途径及其信号通路可否成为DENV感染治疗的新靶点。

The previous studies have shown that the passage HUVEC can be induced autophagy by DENV2, the DEN2 NS1 in the cytoplasm is colocalized with the autophagy marker protein LC3B and the lysosomal marker protein LAMP1, but the mechanism of signaling pathways is unknown.In this study, molecular biology, proteomics and Gene knockdown were used to observe the effects of DENV infection on autophagy of host cells. Using autophagy and its signal pathway inhibitor and stimulator, the dynamics of autophagy on virus replication, release and infection and its effect on host proliferation and function were detected. Through the deep research on the signal path of DENV2 infected HUVEC-induced autophagy AMPK/ERK/mTOR, the target point of DENV2 induced HUVEC apoptosis was found. To construct the target locus gene knockout HUVEC cell line and mice, verified the correlation between HUVEC dysfunction, virus proliferation and autophagy caused by DEVV-2 infection, and the characteristics of immune response in target locus gene knockout mice. The aim is to provide sufficient scientific basis for elucidating the pathogenesis and treatment of DENV infection, and to determine whether autophagy pathway and its signal pathway can become a new target for the treatment of DENV infection.