DC-SIGN受体促进肠道病毒71型体外感染树突状细胞

DC-SIGN molecules promote EV71 infection in dendritic cells in vitro

目的探讨树突状细胞特异性细胞间黏附分子3结合非整合素(dendritic cell specific intercellular adhesion molecule-3 grabbing non integrin,DC-SIGN)的表达对肠道病毒71型(enterovirus type 71,EV71)体外感染人树突状细胞(dendritic cells, DCs)和293T细胞的影响。方法梯度密度离心法分离人外周血单个核细胞(peripheral blood mononuclear cells,PBMCs),体外诱导培养DCs,通过RT-PCR方法克隆DC-SIGN分子,并构建稳定表达DC-SIGN分子的载体pLB-DC-SIGN,脂质体方法转染293T细胞。分别用EV71感染对照组293T细胞和过表达DC-SIGN的293T细胞,荧光定量PCR检测病毒mRNA,蛋白质印迹法检测EV71/VP1的表达水平;同时用DC-SIGN抑制剂酵母甘露聚糖阻断DCs表面的DC-SIGN分子后,荧光定量PCR检测病毒mRNA,蛋白质印迹法检测EV71/VP1的表达水平。结果过表达DC-SIGN的293T细胞与对照组293T细胞相比,感染病毒量明显增高,EV71/VP1表达量显著增加,而阻断DC-SIGN的DCs病毒感染量低于对照组DCs,EV71/VP1表达量明显降低,差异均有统计学意义(P<0.05)。结论 DC-SIGN分子可促进EV71体外感染DCs和293T细胞,可能是EV71感染的受体之一。

Objective To investigate the influence of dendritic cell specific intercellular adhesion molecule-3 grabbing non integrin (DC-SIGN) expression on the infection of enterovirus type 71 (EV71) for dendritic cells (DCs) and 293T cells. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood by gradient density centrifugation and induced to DCs. The DC-SIGN gene was amplified by polymerase chain reaction (PCR) for construction of transient expression vector pLB-DC-SIGN which was then transfected into 293T cells using Lipofectamine TM 2000. After 293T cell and 293T-pLB-DC-SIGN was infected with EV71, EV71 mRNA was identified by fluorescence quantitative PCR. EV71/VP1 was identified by western blot. After the expression of DC-SIGN on DCs was blocked by yeast mannan, EV71 mRNA was identified by fluorescence quantitative PCR and EV71/VP1 was identified by western blot. Results EV71 viral load and EV71/VP1 expression level in 293T cells overexpressed DC-SIGN was significantly higher than that of control 293T, while the viral load and EV71/VP1 expression level in DCs blocked with DC-SIGN inhibitor was lower than that of control DCs ( P <0.05). Conclusion DC-SIGN may be one of the receptors of EV71 who promote EV71 infection in DCs and 293T cells in vitro .