旋毛虫ES抗原致小鼠骨髓源树突状细胞吲哚胺2,3-双加氧酶表达的影响

Effects of Trichinella spiralis ES antigen on expression of indoleamine 2,3-dioxygenase in bone marrow-derived dendritic cells

为探讨旋毛虫排泄分泌(ES)抗原体外诱导小鼠骨髓来源的树突状细胞(DC2.4)在不同时间点对吲哚胺2,3-双加氧酶(IDO)的表达情况,将ES抗原(终浓度为15?g/mL,试验组)、重组小鼠IFN-γ(浓度为1 000 U/mL,阳性对照组)和RPMI 1640培养液(阴性对照组)加入到状态良好的树突状细胞中,并设无添加的空白对照组,分别刺激0、2、6、12、24 h后收集细胞,采用细胞免疫组织化学方法检测各组树突状细胞IDO的表达情况,实时荧光定量法检测各组IDO、IFN-γ、IL-10、TNF-αmRNA表达水平,W estern-blot方法检测各组IDO蛋白表达水平。结果显示,ES抗原、IFN-γ均能提高IDO在细胞质中的mRNA及蛋白表达水平,且与空白对照组呈显著性差异(*P<0.05,*P<0.01,***P<0.001);随着刺激时间的增加,IDO、IFN-γ、IL-10、TNF-αm RNA及IDO蛋白表达量均显著上升,且与空白对照组呈显著性差异(***P<0.001)。结果证实,ES抗原可刺激树突状细胞表达IDO,且存在一定的时间效应,可能与旋毛虫逃避宿主的免疫有一定关系。

To investigate the effect of Trichinella spiralis excretion and secretion(ES) antigen on the expression of indoleamine 2,3-dioxygenase(IDO) in mouse bone marrow-derived dendritic cells(DC2.4) in vitro,the DC 2.4 were cultivated in the presence of various stimuli in four groups:ES antigen(final concentration:15 μg/ml,experimental group),recombinant mouse IFN-γ(final concentration:1000 U/m L,as positive control),RPMI 1640 medium(as negative control) and no addition(as blank control).The cells were collected after 0,2,6,12 and 24 h respectively.The expression of IDO in DC 2.4 was detected by immunohistochemistry.The m RNA expression levels of IDO,IFN-γ,IL-10 and TNF-α were determined by real time RT-PCR.What’s more,the protein expression levels of IDO were examined by Western-blot.In result,the increase of the m RNA and protein expression levels of IDO was observed in the presence of both ES antigen and IFN-γ as compared with the blank control group(*P<0.05,**P<0.01,***P<0.001).With the increasing of stimulation time,the expression of IDO,IFN-γ, IL-10, TNF-α m RNA and IDO protein were significantly increased compared to the blank control group(***P<0.001).The above-mentioned results implied that ES antigen may stimulate the expression of IDO in dendritic cells, and the IDO may have impacts on the immune evasion of the T.spiralis.