登革病毒感染的人脐静脉内皮细胞与人CD4＋ T细胞相互作用对黏附分子和抑炎细胞因子表达的影响

Effects of interaction between dengue virus type 2-infected HUVECs and human CD4＋T cells on the expression of adhesion molecules and immunosuppressive factors

目的 探讨Ⅱ型登革病毒（DENV-2）感染的人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVECs）与CD4＋T细胞的共培养对HUVECs表达细胞间黏附分子（ICAM-1）、血管细胞黏附分子（VCAM-1） mRNA和 CD4＋T细胞表达IL-10、TGF-β1 mRNA产生的影响,以进一步了解DENV感染的免疫学机制.方法 S1P1选择性激动剂CYM-5442处理HUVECs 24 h,用103半数组织培养感染剂量（TCID50）的DENV-2感染HUVECs后与人CD4＋T细胞共培养.于4、8、12、24、48及72 h,分别收集细胞样本,用real-time PCR检测DENV-2非结构蛋白1（NS1）、鞘氨醇激酶1（SPHK1）、ICAM-1、VCAM-1、IL-10、TGF-β1 mRNA表达变化.结果 HUVECs被DENV-2感染后,病毒NS1 mRNA的表达呈一定时效性,在24 h达到高峰.在DENV-2感染的不同实验组中,CYM-5442处理和未处理组,DENV-2 NS1 mRNA的表达均无明显差异.HUVECs被CYM-5442处理和DENV-2感染后,SPHK1 mRNA表达明显上升,差异有统计学意义（P〈0.05）;在DENV-2感染的HUVECs与CD4＋T细胞共培养的情况下,HUVECs ICAM-1和VCAM-1 mRNA高表达,相对HUVECs仅被DENV-2感染组和未处理组差异有统计学意义（P〈0.01）,CD4＋T细胞TGF-β1 mRNA表达无明显差异,IL-10 mRNA表达上调,差异有统计学意义（P〈0.05）.结论 实验证明DENV-2能在HUVECs内复制增殖,CD4＋T细胞可抑制DENV-2增殖;CD4＋T细胞对DENV-2感染的HUVECs产生黏附分子VCAM-1和ICAM-1有明显的促进作用,CD4＋T细胞可促使HUVECs活化,增强炎症反应,这可能与DENV-2感染诱发血管通透性升高有关.DENV-2感染的HUVECs对CD4＋T细胞IL-10 mRNA表达有一定上调作用,对TGF-β1的表达没有明显的影响.

Objective To investigate the effects of interaction between human umbilical vein endothelial cells （HUVECs） which were infected with dengue virus type 2 （DENV-2） and CD4＋T cells on the expression of ICAM-1 （intercellular adhesion molecule 1）,VCAM-1 （vascular cell adhesion molecule 1）,IL-10 and TGF-β1 at mRNA level for further understanding the immunological mechanism of DENV infection.Methods HUVECs were treated with CYM-5442,a selective agonist for sphingosine-1-phosphate receptor 1 （S1P1）,for 24 hours and then infected with 103 TCID50 （50% tissue culture infective dose） of DENV-2 before co-culturing with CD4＋T cells.Changes in the expression of NS1 （DENV-2 nonstructural protein）,SPHK1 （sphingosine kinase 1,phosphorylating sphingosine to S1P）,ICAM-1,VCAM-1,IL-10 and TGF-β1 at mRNA level were detected by real-time PCR after 4,8,12,24,48 and 72 hours of co-culturing.Results There was a certain timeliness in the expression of NS1 at mRNA level after infecting HUVECs with DENV-2 and the expression reached a peak at 24 h.Treating HUVECs with or without CYM-5442 had no significant influence on the expression of DENV-2 NS1 at mRNA level.The expression of SPHK1 at mRNA level was significantly increased after treating HUVECs with CYM-5442 and DENV-2 （P〈0.05）.Compared with DENV-2-infected or untreated HUVECs,Co-culturing DENV-2-infected HUVECs with CD4＋T cells increased the expression of ICAM-1 and VCAM-1 in HUVECs at mRNA level （P〈0.01） as well as the expression of IL-10 in CD4＋T cells at mRNA level （P〈0.05）,but had no significant influence on the expression of TGF-β1 in CD4＋T cells at mRNA level.Conclusion This study shows that DENV-2 can replicate and proliferate in HUVECs,but CD4＋T cells inhibit the replication and proliferation.CD4＋T cells play an important role in promoting the expression of VCAM-1 and ICAM-1 in DENV-2-infected HUVECs at mRNA level,activating HUVECs and increasing inflammation,which may be associated with increased vascular permeability induced by DENV-2 infection.Co-culturing CD4＋T cells with DENV-2-infected HUVECs promotes the expression of IL-10 in CD4＋T cells at mRNA level,but has no significant effect on TGF-β1.