通过大量前期工作，发现DENV感染传代和原代HUVEC后，导致细胞损伤的机制可能不同。设想原代HUVEC受到DENV作用后，可能作为“始作俑者”, 发挥免疫调节作用。计划利用DENV-2，作用原代和传代人HUVEC，经CYM-5442和和特异性抗体作用，与人不同免疫细胞共培养，ELISA法检测培养上清主要炎性细胞因子；q-PCR检测细胞因子mRNA表达水平、凋亡和病毒感染率；流式细胞术动态观察免疫细胞特征性膜表面分子。皮下多点注射建立感染小鼠模型；分选不同免疫细胞，加入相关细胞因子体外培养，观察细胞因子产生情况；动态观测感染小鼠外周血、脾脏CD4+T、CD8+T和Th17细胞的数量和功能；q-PCR检测IL-17、RORγt、IFN-γmRNA的动态表达水平和感染小鼠不同脏器病毒载量；观察组织病理和超微结构变化等，明确HUVEC对DENV感染可能发挥的免疫调节作用及其机制。

Through a great deal of previous researches, we found after DENV was infected into HUVEC passage cell and primary cell, the mechanism of cell damage might be different. Given the fact that DENV immediately resulted in damage of HUVEC passage cell but seemed to have no immediate damage impact on HUVEC primary cell, we supposed that after HUVEC primary cell was attacked by DENV, it might begin to paly a role in immunoregulation. In this research, we plan to use DENV 2 strain to attack HUVEC primary and passage cell, and use CYM-5442 and the specific antibody to act on the cell lines. After the cell lines are cocultured with human immune cells respectively, we will adopt ELISA to detect the level of main inflammatory cytokines in cell culture supernatant. q-PCR method will be adopted to detect mRNA expression level, apoptosis rate and infection rate of different cytokines and flow cytometry will be adopted to dynamically observe the expression of characteristic molecule of membrane surface. By multiple dots subcutaneous injection, we will set up a model of DENV-infected mouse, and define the specific route and time of virus circulating through the body. We also will sort and choose different immunological cells, add into relevant cytokines and culture them in vitro, observe the growth of cytokines, and dynamically observe the quantities and function of infected mouses’ peripheral blood cells, spleen CD4+T, CD8+T and Th17 cells. q-PCR method will be adopted again to detect the dynamic expression level of IL-17、RORγt、IFN-γmRNA and viral load of different organs of infected mouses. We also will observe the histopathology of tissues and organs and ultrastructure changes, thereby define the potential function and mechanism of HUVEC immunoregulation against DENV infection.