登革2型病毒体外诱导Ana-1巨噬细胞极化及其TLR7表达的研究

The research of the polarization and the expression of TLR7 by Ana-1 cell infected with DEN2

目的:研究登革2型病毒体外诱导Ana-1细胞极化及TLR7表达情况,分析其在登革病毒致病过程中的可能作用。方法:用DEN2感染Ana-1细胞,24、72、120小时荧光定量PCR(Real-time PCR)检测病毒核酸、iNOS、Arg-1、IL-10、IL-12p40 mRNA;Western blot检测iNOS、Arg-1、TLR7蛋白;双抗体夹心ELISA法检测TNF-α水平。结果:巨噬细胞被DEN2感染后病毒核酸在72小时达峰值,2-ΔΔCt值为159.98±37.80;iNOS mRNA及蛋白表达明显上升(P<0.05),IL-12p40 mRNA、TNF-α水平显著升高(P<0.05),72小时达峰值;IL-12p40 mRNA 2-ΔΔCt值为11.1±2.41,TNF-α浓度为(454.72±13.41)pg/ml。Arg-1 mRNA及蛋白较M2型极化对照组降低(P<0.05);IL-10 mRNA与正常对照组无显著差异(P<0.05);TLR7表达低于同期正常对照组。结论:Ana-1细胞被DEN2感染后向M1型极化;DEN2可下调Ana-1细胞TLR7表达,可能是DEN2发生免疫逃逸的机制之一;Ana-1细胞TNF-α水平与细胞内DEN2核酸呈正相关。

Objective：To investigate the effect of polarization and the expression of TLR7 in Ana-1 cell which infected with DEN2.Methods： Ana-1 was infected with DEN2 and identified by Real-time PCR and direct immunofluorescence assay.The levels of iNOS,Arg-1,IL-10,IL-12p40 mRNA were detected by Real-time PCR.The protein express of iNOS,Arg-1,TLR7 were analyzed by Western blot.TNF-α in supernatant was examined by ELISA.Results： DEN2 could infected Ana-1 in vitro,which were identified by RT-PCR and direct immunofluorescence assay,the rate of viral replication was increased at 72 h post-infection 2-ΔΔCt was 159.98±37.80（P0.05）;the expression of iNOS mRNA and protein in infected group were significantly increased,which was a marked increase at 72 h（P0.05）.The expression of IL-12p40 mRNA and the concentration of TNF-α were obviously higher（P0.05）.The expression of Arg-1 mRNA and protein in infected group were lower than M2 group（P0.05）;the expression of IL-10 mRNA had few differences between the control group（P0.05）;the protein expression of TLR7 in infected group were decreased.Conclusion： Ana-1 could be infected by DEN2,and induced polarization to M1.The lower expression of TLR7 in Ana-1 may be one of the mechanisms of DEN2 escaping immune response;the level of TNF-α were positively correlated with viral load.