DEN2感染对小鼠髓源性树突状细胞膜表面分子TLR4、TLR7表达的影响

Dengue virus type 2 infection inhibits the expression of toll-like receptors 4 and 7 in murine bone marrow-derived dendritic cells

目的探讨登革病毒2型（DEN2）感染对小鼠髓源性树突状细胞表面分子TLR4、TLR7表达影响。方法BALB／C乳鼠脑内接种及C6／36细胞增殖病毒，RT．PCR鉴定病毒核酸；rmGM．CSF和rmIL-4联合诱导鼠源性DC；直接免疫荧光法观察DEN2感染DC；流式细胞术检测DC被DEN2感染后膜表面分子CD11C、CD86、I-A／I．E类分子表达的动态变化；RT—PCR动态检测DEN2感染DC后DEN2RNA、TLR4和TLR7mRNA水平表达的变化。结果IL_4和GM．CSF诱导小鼠骨髓来源DC的纯度可达到70％；DEN2能够感染小鼠骨髓来源DC；与阴性对照组相比，接种病毒组[1×104半数组织培养感染剂量（TCID50）]的DC膜表面分子CD86、I-A／I．E的百分率差异无统计学意义。与阴性对照组相比，接种病毒组（1×105 TCID50）的DCs膜表面分子CD86、I-A／I—E的百分率差异均有统计学意义．但各组百分率的变化并未随着时间的延长呈现出明显的上升或下降的趋势；随着病毒剂量的增加，DC膜表面分子CD86、I-A／I—E的百分率呈现出上升的趋势；与阴性对照组比较，RT．PCR证明各组病毒mRNA水平随着病毒剂量的增大而逐渐升高。与阴性对照组比较，RT—PCR检测各组被DEN2感染的DCTLR4、TLR7mRNA随着病毒剂量的增加，呈现下调的趋势。结论DEN2可促进DC的成熟；DEN2感染DC后，TLR4、TLR7mRNA水平随病毒mRNA水平的增加而降低，提示TLR4、TLR7与病毒感染密切相关，在DEN致病中发挥了一定的作用和功能。

Objective To analyze the changes in the expression of Toll-like receptors 4 （TLR4） and 7 on the surface of murine bone marrow-derived dendritic cells following dengue virus type 2 （ DEN2 ） infection. Methods DEN2 NGC strain was infected into BALB/c suckling mice through intracranial injec- tion and injected into C6/36 cells to induce the in vivo and in vitro proliferation of DEN2, respectively. RT- PCR was performed to identify DEN2 RNA. Reed-Muench method was used to determine the 50% tissue culture infective dose （ TCIDs0 ） of DEN2. Dendritic cells （DCs） were prepared by stimulating bone marrow cells isolated form C57BL/6 mice with IL-4 and GM-CSF and then identified by flow cytometry. The pre- pared murine bone marrow-derived DCs were infected with DEN2 and observed with direct immunofluores- cence assay. Dynamic changes in the expression of CDllc, CD86 and I-A/I-E molecules on DCs after DEN2 infection were detected by flow cytometry. Levels of DEN2 RNA and the expression of TLR4 and TLR7 at mRNA level were dynamically detected by real-time quantitative PCR. Results The TCIDs0 of DEN2 to C6/36 cells was 10-5＇s. Murine bone marrow-derived DCs were acquired with a purity of 70% and could be infected with DEN2 in vitro. The percentages of CD86 and I-A/I-E molecules on the surface of DCs infected with 1 ×105 TCID50 of DEN2 were statistically different from those of the negative control group. Nei-ther of the two groups showed a significant difference in the percentages of membrane molecules over time. However, the percentages of membrane molecules on DCs increased with increasing viral load. Compared with the negative control group, the levels of DEN2 RNA in infection groups were increased with increasing virus load, while the expression of TLR4 and TLR7 on DEN2 infected-DCs at mRNA level was decreased with increasing viral load. Conclusion DEN2 infection promotes the maturation of DCs. Expression of TLR4 and TLR7 on DEN2 infected-DCs at mRNA level decreases with increasing viral load, which suggests that TLR4 and TLR7 are closely related to viral infection and play a certain role in the pathogenesis of DEN infection.