DC-SIGN受体促进肠道病毒71型体外感染树突状细胞
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摘要
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 293 T 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 293 T cells using Lipofectamine~(TM) 2000. After 293 T cell and 293 T-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 293 T cells overexpressed DC-SIGN was significantly higher than that of control 293 T, 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 293 T cells in vitro.
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 293 T 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 293 T cells using Lipofectamine~(TM) 2000. After 293 T cell and 293 T-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 293 T cells overexpressed DC-SIGN was significantly higher than that of control 293 T, 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 293 T cells in vitro.
引文
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[3]Emara M,Royer PJ,Mahdavi J,et al.Retagging identifies dendritic cell-specific intercellular adhesion molecule-3 (ICAM3)-grabbing non-integrin (DC-SIGN) protein as a novel receptor for a major allergen from house dust mite[J].J Biol Chem,2011,287(8):5756-5763.
[4]Gringhuis S,den Dunnen J,Litjens M,et al.Carbohydrate-specific signaling through the DC-SIGN signalosome tailors immunity to Mycobacterium tuberculosis,HIV-1 and Helicobacter pylori[J].Nat Immunol,2009,10(10):1081-1088.
[5]Van BW,P?hlmann S,Favoreel HW,et al.Bitter-sweet symphony:glycan-lectin interactions in virus biology[J].FEMS Microbiol Rev,2014,38(4):598-632.
[6]de Jong MA,de Witte L,Bolmstedt A,et al.Dendritic cells mediate herpes simplex virus infection and transmission through the C-type lectin DC-SIGN[J].J Gen Virol,2008,89(Pt10):2398-2409.
[7]Lin YW,Wang SW,Tung YY,et al.Enterovirus 71 infection of human dendritic cells[J].Exp Biol Med(Maywood),2009,234(10):1166-1173.
[8]Zhang F,Ren S,Zuo Y.DC-SIGN,DC-SIGNR and LSECtin:C-type lectins for infection[J].Int Rev Immunol,2014,33(1):54-66.
[9]Feinberg H,Guo Y,Mitchell DA,et al.Extended neck regions stabilize tetramers of the receptors DC-SIGN and DC-SIGNR[J].J Biol Chem,2005,280(2):1327-1335.
[10]Tacken PJ,Ginter W,Berod L,et al.Targeting DC-SIGN via its neck region leads to prolonged antigen residence in early endosomes,delayed lysosomal degradation,and cross-presentation[J].Blood,2011,118(15):4111-4119.