DENV-2 感染的HUVECs 与CD4+ T 细胞相互作用对炎性细胞因子产生的影响

目的:研究原代人脐静脉内皮细胞(Primary human umbilical vein endothelial cells,HUVEC)被登革域型病毒 (DENV鄄2)感染,与CD4+ T 细胞共培养后,对产生主要炎性细胞因子的相互影响。方法:密度梯度离心法提取浓缩白细胞中的 PBMC,免疫磁珠法阴性分选CD4+ T 细胞,流式检测细胞表面CD3,CD4 分子的表达和CD4+ T 细胞的纯度。HUVECs 经S1P1 特异性受体激动剂CYM-5442 预处理24 h,加入103 TCID50 的DENV-2 感染后,与CD4+ T 细胞共培养,Real鄄time RT-PCR 动态 检测DENV-2 感染HUVECs 后病毒NS1 基因及IL-6、IL-8 的mRNA 和CD4+ T 细胞内IL-4、IL-17、TNF-α、IFN-β的mRNA 相对 表达量;双抗体夹心ELISA 法检测培养上清IL-6、IL-8 的表达。结果:流式检测经免疫磁珠阴选的CD4+ T 细胞纯度为(98.02± 0.32)%。DENV-2 感染HUVECs 后病毒NS1 基因相对表达逐渐增加,在24 h(3.03±0.26,P<0.001)达到峰值后下降,而感染 DENV-2 后与CD4+ T 细胞共培养组的NS1 基因相对表达量低于感染组,且呈下降趋势。感染后IL-6 和IL-8 表达均有上调,与 CD4+ T 细胞共培养后,IL-6 和IL-8 在各时间点表达均明显升高(P<0.01)。CYM-5442 预处理的共培养感染组中,IL-6 在24 h (28.91±2.34,P<0.05)、36 h(19.36依0.1,P<0.05)和72 h(13.84±0.82,P<0.05)显著下降,IL-8 表达显著下降。与感染后的 HUVEC 共培养后CD4+ T 细胞的IL-4、IL-17、TNF-α、IFN-β的mRNA 表达均明显升高。结论:DENV-2 能感染原代HUVECs;与 CD4+ T 细胞共培养后NS1 的表达被抑制。CD4+ T 细胞不仅能增强被DENV-2 感染的HUVEC 的活化,同时也能被感染后的 HUVEC 活化。

Influence of interaction between CD4+ T cells and human umbilical vein#br# endothelial cells infected with DWNV-2 on production of inflammatory cytokines

Objective:To study the interaction of the inflammatory cytokines expression between CD4+ T cells and primary human umbilical vein endothelial cells (HUVECs) infected by dengue virus (DENV-2).Methods:PBMC was extracted from white blood cells by density gradient centrifugation,CD4+ T cells were sorted by immunomagnetic beads.The expression of CD3 and CD4 molecules on the surface of cells was detected by flow cytometry to identify the purity of CD4+ T cells.First,HUVECs were pretreated by specific-S1P1 receptor agonist CYM-5442 for 24 h,second,infected by DENV-2 on the titer of 103 TCID50,then co-culturing with CD4+ T cells,The relative expression of NS1 partial sequence and IL-6,L-8 mRNA of HUVEC,and IL-4,IL-17，TNF-α，IFN-β of CD4+ T cells detected by Real.time RT-PCR.IL-6 and IL-8 secreted in cultured supernatant analyzed by ELISA.Results:The purity of CD4+ T cells was (98.02±0.32)%.The expression of NS1 gradually increased to 24 h (3.03±0.26,P<0.001),decreased after reaching the peak.The relative expression of NS1 in the group of co-cultured with CD4+ T cells was lower than other groups.After infection,the expression of IL- and IL-8 were up-regulated,and the expression of IL-6 and IL-8 at each time point was significantly increased after co-culturing with CD4+ T (P<0.01).IL-6 of CYM-5442 pretreatment group,in 24 h (28.91±2.34,P<0.05),36 h(19.36±0.1,P< 0.05) and 72 h(13.84±0.82,P<0.05) was significantly decreased,the expression of IL-8 also decreased significantly.The mRNA ex- pression of IL-4，IL-17，TNF-α and IFN-β in CD4+ T cells was significantly increased after co-culturing with HUVECs.After the treatment with CYM-5442 group,the expression was decreased.Conclusion:DENV-2 could infect the primary HUVECs,and the expression of NS1 was inhibited after co-culturing with CD4+ T cells.CD4+ T cells can not only enhance the activation of HUVECs infected by DENV-2,but also can be activated by the infected HUVECs infected with DENV-2.