摘要
目的检测不同的棘球蚴抗原体外诱导树突状细胞(DCs)表达吲哚胺2,3-双加氧酶(IDO)的情况。方法从C57BL/6小鼠股骨中分离出骨髓细胞,用小鼠重组巨噬细胞集落刺激因子(rmGM-CSF)诱导后,获得小鼠骨髓源树突状细胞(BMDCs)。分别用重组抗原B(rAgB,15μg/ml)、小鼠细粒棘球蚴囊液(MHF,5 mg/ml)、γ干扰素(IFN-γ,1 000 U/ml,为阳性对照)和RPMI 1640完全培养液(阴性对照)刺激DCs,于18、24和48 h后收集细胞和细胞上清。采用流式细胞术检测各组DCs的表面标志物CD40、CD80、CD86和I-A/I-E的阳性表达情况,并利用实时荧光定量PCR(FQ-RT-PCR)检测各组的IDO mRNA相对转录水平。利用高效液相色谱法(HPLC)检测各组细胞上清中的色氨酸(Try)浓度。结果流式细胞术检测结果显示,DCs受rAgB和MHF刺激后,其表面标志物CD40、CD80、CD86和I-A/I-E的阳性表达率均降低。刺激24 h后,rAgB组的CD40、CD86和I-A/I-E阳性表达率分别为(22.60±2.69)%、(35.50±4.38)%和(57.30±4.38)%,与MHF组[(38.00±3.54)%、(53.00±3.39)%和(77.10±1.70)%]和阴性对照[(37.95±3.61)%、(19.55±1.06)%和(85.45±1.63)%]的差异均有统计学意义(P<0.05)。FQ-RT-PCR结果显示,刺激18、24和48 h后,rAgB组的IDO mRNA水平[(9.20±0.01)、(29.44±0.02)和(16.48±0.04)]和MHF组的[(9.67±0.02)、(17.52±0.01)和(16.81±0.01)]均高于阴性对照组[(2.46±0.01)、(7.77±0.01)和(10.56±0.01)](P<0.01),rAgB组和MHF组IDO mRNA水平的差异均有统计学意义(P<0.05)。HPLC结果显示,刺激18、24和48 h后,rAgB组DCs上清中的色氨酸浓度[(23.65±0.64)、(13.95±1.06)和(19.05±0.64)μmol/L]均较其他3组低,刺激24 h后的色氨酸浓度与阴性对照组(22.90±0.14)和MHF组(20.65±0.35)的差异均有统计学意义(P<0.05)。结论在体外实验条件下,rAgB、MHF均可上调DCs表面IDO的表达,作用24 h后,rAgB上调IDO的能力强于MHF。
Objective To observe the expression of indoleamine 2, 3-dioxygenase （IDO） in dendritic cells （DCs） via different Echinococcus granulosus antigens in vitro. Methods Bone Marrow DCs generated from bone marrow precursor cells of C57BL/6 mice and cultured in the presence of recombinant mouse GM-CSF （rmGM-CSF）. Then, DCs were induced with 15 μg/ml recombinant antigen B （rAgB）, 5 mg/ml mouse hydatid fluid （MHF）, 1 000 U/ml IFN-γ （as positive control）, and RPMI 1640 complete medium （as negative control）, respectively. Meanwhile, the treated DCs and cell supernatants were collected at 18, 24 and 48 h after induction. The positive expressions of D40, CD80, CD86 and I-MI-E on DCs were determined by flow cytometry. By real-time fluorescent quantitative reverse-transcription polymerase chain reaction （FQ-RT-PCR）, the expression level of 1DO mRNA in DCs was measured. Concentrations of tryptophan （Try） were tested by high-performance liquid chromatography （HPLC） assay in cell supernatant. Results The data from flow cytometry showed that the positive expressions of CD40, CD80, CD86, I-A/I-E were decreased after stimulated by rAgB and MHF. At 24 h after induction, there was significant difference in the level of CD40, CD86 and I-A/I-E among rAgB-treated group [（22.60±2.69）%, （35.50±4.38）%, （57.30±4.38）%], MHF-treated group [（38.00±.54）%, （53.00± 3.39）%, （77.10±1.70）%1 and negative control [（37.95±3.61） %, （19.55±1.06） % and （85.45±1.63） %（P〈0.05）. At 18, 24 and 48 h after induction, the levels of IDO mRNA in rAgB-treated group [（9.20±0.01）, （29.44±0.02）, （16.48± 0.04）] and MHF-treated group [（9.67±0.02）, （17.52±0.01）, （16.81±0.01）] was higher than that of negative control group[（2.46±0.01）, （7.77±0.01）, and（10.56-＋0.01）l（P〈0.01）. And significant difference was found between rAgB-treated group and MHF-treated group （P〈0.05）. At 18, 24 and 48 h after induction, the concentrations of
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2013年第3期188-192,共5页
Chinese Journal of Parasitology and Parasitic Diseases
基金
新疆维吾尔自治区自然科学基金(No.200821132)
国家自然科学基金(No.30800967)~~
