DENV-2作用于HUVECs和人巨噬细胞对黏附分子表达的影响

Effects of DENV-2-infected HUVECs and macrophages on adhesion molecules

目的:研究登革Ⅱ型病毒(DENV-2)感染人脐静脉内皮细胞(HUVECs)和人巨噬细胞,且感染后共培养对主要黏附分子表达的影响。方法:水平密度梯度离心法提取浓缩白细胞中的外周血单核细胞(PBMC),贴壁分离单核细胞,用巨噬细胞集落刺激因子(M-CSF)刺激5~7 d,流式细胞术鉴定细胞表面CD14和CD11b分子。用10 3 TCID50 DENV-2分别感染HUVECs和人巨噬细胞,且感染后两者共培养,qRT-PCR检测细胞内不同时间点DENV NS1、VCAM-1、ICAM-1和E-selectin mRNA相对表达量。双抗体夹心ELISA法分别检测不同时间点各组培养上清液中可溶性分子ICAM-1、VCAM-1和E-selectin的表达水平变化。结果:流式细胞术鉴定人巨噬细胞纯度为(92.15±1.24)%。DENV-2感染HUVECs后病毒基因NS1的相对表达量呈先升后降趋势,在24 h达到峰值后下降,DENV-2感染人巨噬细胞的DENV NS1基因相对表达量呈先递减后增加趋势,共培养组HUVECs的NS1 mRNA各时间点表达水平均高于HUVECs单独感染组。DENV-2感染巨噬细胞后,ICAM-1 mRNA相对表达量于4、8 h高于巨噬细胞未感染组,12、24、48、72 h相对表达水平与未感染组相比差异无统计学意义;E-selectin mRNA相对表达量于4 h明显高于巨噬细胞未感染组,8 h与未感染组相比表达量无统计学意义差异,12、24、48、72 h与未感染组相比表达量下调;但未检测到VCAM-1 mRNA的表达。共培养组HUVECs的ICAM-1和VCAM-1 mRNA相对表达量与HUVECs单独感染组相比8 h后均上调,且均在24 h达峰值。感染后的HUVECs E-selectin mRNA于4 h表达水平最高,且各组相对表达量均低于共培养组。共培养组中感染DENV-2的HUVECs培养上清液中ICAM-1表达量均高于HUVECs单独感染组;共培养组中感染DENV-2的HUVECs中E-selectin表达量随时间呈先增后降趋势,且在24 h表达量达峰值;但未检测到培养上清液中VCAM-1的表达。结论: DENV-2能在HUVECs和人巨噬细胞中复制且能激活内皮细胞,被DENV-2感染的巨噬细胞能增强被感染的HUVECs的细胞活化并增加其黏附分子的表达。

Objective: To study the effects of dengue virusⅡ(DENV-2)-infected human umbilical vein endothelial cells (HUVECs) and macrophages on adhesion molecules expression. Methods: Monocytes were extracted by horizontal density gradient centrifugation and stimulated with M-CSF factors for 5-7 days.CD14 and CD11b molecules on cells surface were identified by flow cytometry.HUVECs and human macrophages were infected with 10 3TCID50 DENV-2,then infected-cells were co-cultured.The relative expression of DENV NS1,VCAM-1,ICAM-1 and E-selectin mRNA was detected by qRT-PCR.The secretion of VCAM-1,ICAM-1 and E-selectin in culture supernatant was analyzed at different time by ELISA. Results: The purity of macrophages was (92.15±1.24)%.The expression of DENV-2 NS1 mRNA in HUVECs increased and then reached the peaking at 24 h,in macrophages decreased first then increased.The expression of NS1 mRNA in HUVECs of co-culture group was higher than infected-HUVECs group.In DENV-2-infected macrophages,the relative expression of ICAM-1 mRNA was higher than that in uninfected group at 4 h and 8 h,there was no significant difference between 12 h,24 h,48 h and 72 h.The relative expression of E-selectin mRNA was significantly higher than that in uninfected-macrophages group at 4 h,and no significant difference in 8 h.The expression level was down-regulated at 12 h,24 h,48 h and 72 h compared with uninfected group,but VCAM-1 mRNA was not detected.The relative expression of ICAM-1 and VCAM-1 mRNA in HUVECs of co-culture group was up-regulated after 8 h and higher than that in infected-HUVECs alone,and peaked at 24 h.The E-selectin mRNA in HUVECs was the highest at 4 h,but the relative expression in each group was lower than that in the co-culture group.The secretion of ICAM-1 in the co-culture supernatant of DENV-2-infected HUVECs was higher than that in the independent infection group of HUVECs.E-selectin in the co-culture group increased first then decreased,and peaked at 24 h,but the secretion of VCAM-1 in supernatant was not detected. Conclusion: DENV-2 could replicate in HUVECs and human macrophages and activate HUVECs.DENV-2-infected macrophages could enhance and increase expression of adhesion molecules of DENV-2-infected HUVECs.