中文摘要：

目的：研究原代人脐静脉内皮细胞（Primary human umbilical vein endothelial cells,HUVECs）被登革Ⅱ型病毒（DENV-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10^3TCID50的DENV-2感染后,与CD4^＋T细胞共培养,Real-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-γ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±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HUVECs共培养后CD4^＋T细胞的IL-4、IL-17、TNF-α、IFN-γ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HUVECs的活化,同时也能被感染后的HUVECs活化。

英文摘要：

Objective：To study the interaction of the inflammatory eytokines 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 eentrifugation, CD4＋ T cells were sorted by immunomaguetic 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-S1 P1 receptor agonist CYM-5442 for 24 h, second, infected by DENV-2 on the titer of 103 TCID50, then eo-cuhuring with CD4＋ T cells,The relative expression of NS1 partial sequence and IL-6,L-8 mRNA of HUVECs,and IL-4,IL-17,TNF-α,IFN-γ 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-6 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-γ 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 act