肠道病毒71型诱导人脑微血管内皮细胞的凋亡

Enterovirus 71 induces apoptosis in human brain microvascular endothelial cells

目的探讨肠道病毒71型(enterovirus 71,EV71)诱导人脑微血管内皮细胞(human brain microvascular endothelial cells,HBMECs)的凋亡及其相关机制。方法流式细胞仪检测EV71能否诱导HBMECs的凋亡;免疫荧光方法观察EV71诱导HBMECs线粒体膜电位的改变情况;免疫印迹检测DJ-1蛋白在EV71感染HBMECs不同时间点的表达;酶联免疫方法检测EV71感染HBMECs不同时间点TNF-α、IL-10、IL-4的分泌情况。结果 EV71感染HBMECs不同时间点的HBMECs的早期凋亡数与晚期凋亡、坏死细胞数具有显著差异(P<0.01);随着感染时间延长,HBMECs早期凋亡数与晚期凋亡、坏死细胞数均有所增加,但以晚期凋亡、坏死细胞数增加更明显(P<0.01)。与对照组相比,EV71感染HBMECs8 h线粒体膜电位明显降低(P<0.01);EV71感染HBMECs 16、24 h,DJ-1表达增强(P<0.05)。EV71感染HBMECs不同时间点TNF-α、IL-10、IL-4的浓度具有明显差异(P<0.01)。结论 EV71能诱导HBMECs的凋亡以及改变线粒体膜通透性,EV71还能诱导HBMECs中DJ-1的表达改变和TNF-α、IL-10、IL-4的分泌改变。

Objective To investigate whether enterovirus 71 （EV71） induces the apoptosis in human brain microvascular endothelial cells （HBMECs） and the possible underlying mechanism. Methods The apoptosis of HBMECs was detected by flow cytometry after EV71 infection. The mitochondrial membrane potential of the infected HBMECs was detected by immunofluorescence staining. The expression of DJ-1 in the infected HBMECs was observed by Western blot analysis. Concentration of TNF-α, IL-4 and IL-10 in the supernatant of HBMECs was detected by ELISA. Results EV71 induced apoptosis in HBMECs in a time-dependent fashion （P〈0.01）. With the elapse of infection time, the numbers of terminal apoptosis cells and necrotic cells were increased significantly, compared with the cells at the early apoptosis （P〈0.01）. EV71 infected HBMECs resulted in significant decreased in the mitochondrial membrane potential in 8 h after infection than the control （P〈0.01）. Higher expression of DJ-1 protein was observed in 16 and 24 h after EV71 infection when compared with the control （P〈0.05）. Significant differences were found in the supernatant concentrations of TNF-α, IL-4, and IL-10 in EV71 infected HBMECs for different time periods （P〈0.01）. Conclusion EV71 induces the apoptosis and loss of Δψm of HBMECs, and the expression of DJ-1 and the releases of TNF-α, IL-4, and IL-10 in HBMECs.