HUVEC被登革病毒感染后免疫调节作用的研究

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.

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。

较好地完成计划。包括原代HUVEC的分离、传代和鉴定，DENV-2 毒株扩增鉴定、毒力检测。DENV-2感染的HUVEC和Balb/c小鼠受CYM-5442干预后免疫机制研究; DENV-2感染后血管内皮细胞和人原代真皮微血管内皮细胞通透性改变机制的研究; DENV-2 感染的HUVECs与调节性T细胞相互作用对主要炎性细胞因子产生的影响和诱导HUVEC自噬和影响细胞活力的研究; DENV-2 感染HUVECs与人巨噬细胞相互作用对主要炎性细胞因子产生和主要粘附分子表达的影响。结果证明HUVECs被DENV2感染后与不同免疫细胞共培养，受CYM-5442作用，炎性细胞因子产生有显著变化。被感染的HUVECs能促使Treg对抑炎性细胞因子的分泌上调，通过产生细胞因子发挥免疫调节作用。建立被DENV-2感染的Balb/c小鼠模型，CYM-5442的干预显著下调各种炎性因子的表达，保护靶器官损伤;DENV感染PHUVEC时，IFNLR1的存在有助于抵抗因病毒作用导致的细胞通透性增加。IFNLR1抵抗DENV感染所致PHUVEC通透性增强效应与其调控下游RhoA激酶活性密切相关。DENVⅡ感染后PHUVEC处于明显活化状态，与IFN-β大量分泌密切相关。DENV-2感染pHDMECs后通透性增加与IL-6和IL-8明显上调关系密切。DENV-2感染的HUVECs能增强Treg IL-10与TGF-β的分泌，产生的抑制性细胞因子能降低被感染的HUVECs炎性细胞因子的产生。DENV-2感染诱导自噬对HUVEC起保护作用，被DENV-2感染的HUVEC能够活化Mø并促使其分泌大量的IL-6、IL-8、TNF-α、IL-1β，被DENV-2感染激活的巨噬细胞能增强被感染的HUVECs的活化并增加其粘附分子的表达。. 本项目培养博士生2人，硕士生4人。发表研究论文6篇，其中SCI收录2篇，国内核心期刊4篇。