登革病毒Ⅱ型引起原代HDMECs通透性变化机制的初步研究

Primary mechanism of changing permeability in DENV-2 infected primary human dermal micro-vascular endothelial cells

目的:了解登革病毒Ⅱ型(Dengue virus Type 2,DENV-2)感染原代人真皮微血管内皮细胞(Primary Human dermal micro-vascular endothelial cells,p HDMECs)引起细胞通透性改变的机制。方法:用103TCID50的DENV-2感染p HDMECs;于4、8、12、24、48 h Real time-PCR、免疫荧光法及流式细胞术检测DENV-2 NS1部分序列及蛋白;Transwell法检测细胞通透性;Real time-PCR和双抗体夹心ELISA法检测IL-6和IL-8的变化;流式细胞术检测24、48、72 h细胞凋亡。结果:DENV-2感染的p HDMECs病毒NS1基因相对表达上调,但未检测到病毒NS1蛋白;DENV-2感染的p HDMECs通透性在24、48 h显著升高;p HDMECs被感染72 h后凋亡也无明显变化;IL-6和IL-8 mRNA分别在8、24 h相对表达上调[IL-6:(2.49±0.5)倍,P<0.05;IL-8:(6.82±1.69)倍,P<0.05];对照组和感染组分泌的IL-6于8 h分别为(869.6±50.7)、(1 248.8±86.9)pg/ml,P<0.05;IL-8于48 h分别为(967.6±156.6)、(1 331.0±86.3)pg/ml,P<0.05。结论:DENV-2能感染p HDMECs;p HDMECs被DENV-2感染后,细胞的通透性增加与凋亡无关,与促炎性细胞因子IL-6和IL-8明显上调关系密切。

Objective： To reveal the primary mechanism of changing permeability in DENV-2 infected p HDMECs. Methods：p HDMECs was incubated by DENV-2 on the concentration of 103TCID50,and the penetrability of the cell was detected by Transwell at4,8,12,24,48 h,respectively. Then,the partial sequence of DENV-2 NS1 was analyzed by Real time-PCR,and NS1 protein was detected by immunofluorescence and flow cytometer（ FCM）. The apoptosis rate of p HDMECs was assayed by FCM. Finally,IL-6 and IL-8 secreted by p HDMECs were analyzed by Real time-PCR and double antibody sandwich ELISA. Results： The relative expression of NS1 gene was elevated but NS1 protein was not detected; the permeability of DENV-2 infected p HDMECs had dramatically increased both at24,48 h,but the apoptosis rate has little changed even been influenced by DENV-2 at 72 h. However,the relative expression of IL-6 / IL-8 mRNA was boosted at 8,24 h[（ 2. 49 ± 0. 50） and（ 6. 82 ± 1. 69） fold,respectively,P〈0. 05]. In protein level,compared with control（ 869. 6 ± 50. 70） pg / ml,IL-6 secreted by DENV-2 infected p HDMECs could reach by（ 1 248. 8 ± 86. 9） pg / ml（ P〈0. 05）,and IL-8 was（ 1 331. 0 ± 86. 3） pg / ml（ P〈0. 05） while the control was（ 967. 6 ± 156. 6） pg / ml. Conclusion： Indeed,p HDMECs can be infected by DENV-2; the increasing permeability of DENV-2 infected p HDMECs may not be caused by the p HDMECs＇ apoptosis but the enhancing of pro-inflammatory cytokine IL-6 / IL-8.