摘要目的 观察原代人脐静脉内皮细胞(primary human umbilical vein endothelial cells,HUVECs)被登革热2型病毒(dengue virus 2,DENV-2)感染后,与人外周血巨噬细胞(macrophages,M?)共培养,两种细胞主要炎性因子产生的变化.方法 密度梯度离心法从人体外周血浓缩白细胞中分离单个核细胞(peripheral blood mononuclear cell,PBMC),贴壁得到单核细胞,通过巨噬细胞集落刺激因子(M-CSF)刺激,流式细胞术检测CD14+CD11b+细胞纯度,得到M?.DENV-2感染HUVECs,real-time PCR检测HUVECs细胞内病毒NS1 mRNA表达量变化.HUVECs被DENV-2感染后,通过Transwell法与M?共培养,对照组用鞘氨醇-1-磷酸(sphingosine-1-phosphate,S1P)1型特异性受体激动剂CYM-5442预处理24 h去除药物后感染病毒.Real-time PCR分别检测HUVECs产生的IL-6、IL-8 mRNA和M?产生的IL-6、IL-8、TNF-α、IL-1βmRNA的逆转录水平;ELISA双抗体夹心法检测培养上清中上述细胞因子的表达.结果 HUVECs DENV-2被感染后,细胞内NS1基因转录水平逐渐上升,24 h时达峰值(2.66±0.53,P<0.05)后下降.流式细胞术检测M?纯度为(89.16±2.07)％.HUVECs被DENV-2感染后IL-6、IL-8 mRNA转录水平均上调,24 h mRNA转录量达到峰值;IL-6为16.10±0.17,IL-8为29.76±0.58.未感染组转录水平IL-6为1.46±0.67,IL-8为1.60±0.54.M?被感染后IL-6、IL-8、TNF-α、IL-1βmRNA表达量均有明显上升,在24 h mRNA IL-6表达量为45.82±3.72,IL-8为52.34±1.69(12 h),TNF-α为28.94±1.75,IL-1β为30.96±1.44;未感染组转录水平IL-6为1.16±0.22,IL-8为1.15±0.21,TNF-α为1.11±0.09,IL-1β为1.47±0.31.HUVECs与M?共培养后上述细胞因子在各时间点的转录水平有所下降,依然明显高于未感染对照组.CYM-5442预处理后,共培养组感染的HUVECs IL-6、IL-8的mRNA转录水平明显下降(P<0.01),M?中IL-6、IL-8、TNF-α、IL-1βmRNA转录水平也有下调(P<0.01).结论 DENV-2能够感染原代HUVECs,且被DENV-2感染的HUVECs能够活化M?并促使其分泌大量的IL-6、IL-8、TNF-α、IL-1β,同时活化的M?能一定程度地降低HUVECs炎症细胞因子的产生.

abstractsObjective To study the influences on the production of major inflammatory cytokines after co-culturing macrophages with human umbilical vein endothelial cells ( HUVECs) that were infected with dengue virus type 2 (DENV-2). Methods Density gradient centrifugation was used to isolate periph-eral blood mononuclear cells ( PBMC) from concentrated human leukocytes. Adherent monocytes in culture flasks were obtained and stimulated with macrophage colony-stimulating factor ( M-CSF) to prepare macro-phages. The purity of CD14+CD11b+ cells was measured by flow cytometry. Changes in the expression of NS1 at mRNA level in HUVECs were detected by real-time PCR following DENV-2 infection. DENV-2-in-fected HUVECs were co-culture with macrophages in Transwell chambers. A control group was set up by pre-treating HUVECs with sphingosine-1-phosphate (S1P) type 1 (S1P1)-specific receptor agonist CYM-5442 for 24 h to remove the drug before infection and then co-culturing the infected cells with macrophages. Real-time PCR was used to detect the expression at mRNA level of IL-6 and IL-8 in HUVECs and IL-6, IL-8, TNF-α and IL-1β in macrophages. A double-antibody sandwich ELISA was used to detect the expression of above cytokines in culture supernatants. Results After HUVECs were infected with DENV-2, expression of NS1 gene at mRNA level gradually increased to the peak at 24 h (2. 66±0. 53, P<0. 05) and then de-creased. The purity of macrophages detected by flow cytometry was (89. 16±2. 07) ％. Expression of IL-6 and IL-8 at mRNA level in DENV-2-infected HUVECs was up-regulated. The peak values reached at 24 h of IL-6 and IL-8 expression were 16. 10±0. 17 and 29. 76±0. 58, while the expression levels at 24 h in the un-infected group were 1. 46±0. 67 and 1. 60±0. 54, respectively. Expression of IL-6, IL-8, TNF-αand IL-1βat mRNA level in DENV-2-infected macrophages was increased significantly. The levels of IL-6, IL-8, TNF-αand IL-1β expression at 24 h were 45. 82±3. 72, 52. 34±1. 69 (12 h), 8. 94±1. 75 and 30. 96±1. 44 in the infected macrophages, and 1. 16±0. 22, 1. 15±0. 21, 1. 11±0. 09 and 1. 47±0. 31 in the uninfected group. Expression of these cytokines was decreased at every time points after co-culturing of DENV-2-infec-ted HUVECs with macrophages, but still significantly higher than that in the uninfected group. In the co-cul-ture group with DENV-2 infection, CYM-5442 pretreatment significantly decreased the expression at mRNA level of IL-6 and IL-8 in HUVECs (P<0. 01) and that of IL-6, IL-8, TNF-αand IL-1βin macrophages (P<0. 01). Conclusions DENV-2 could infect primary HUVECs, and then activate macrophages to promote the secretion of large amounts of IL-6, IL-8, TNF-αand IL-1β. Moreover, the activated macrophages could reduce the production of inflammatory cytokines in HUVECs to a certain extent.