吲哚胺2,3-双加氧酶基因转染未成熟树突细胞对卵蛋白致敏小鼠模型CD4~＋ T细胞的影响

Effects of transfecting immature dendritic cells with indoleamine 2,3-dioxygenase gene on the spleen CD4^+ T cells in ovalbumin-allergized mouse model

目的以pEGFP-N1-IDO真核表达载体转染未成熟树突细胞,观察吲哚胺2,3-双加氧酶(IDO)基因对CD4+ T细胞的影响。方法体外扩增小鼠骨髓来源的未成熟树突细胞,采用光镜、扫描电镜、透射电镜、流式细胞技术鉴定后,采用DOTAP脂质体转染法进行转染,倒置荧光显微镜观察绿色荧光蛋白的表达。同时建立卵蛋白致敏小鼠模型,分离并纯化脾脏CD4+ T细胞,用3H-TdR掺入法进行混合淋巴细胞反应实验,观察IDO转染未成熟树突细胞对卵蛋白致敏小鼠模型脾脏CD4+ T细胞增殖的影响,并用TUNEL法检测CD4+T细胞凋亡情况。结果IDO基因转染未成熟树突细胞能抑制卵蛋白致敏小鼠模型CD4+ T细胞增殖,pEG-FP-N1-IDO质粒转染组CD4+ T细胞减少,与pEGFP-N1空质粒转染组和对照组比较差异显著(P<0.05);IDO基因转染未成熟树突细胞可诱导CD4+ T细胞凋亡,pEGFP-N1-IDO质粒转染组CD4+ T细胞凋亡率为15.3%±2.6%,明显高于对照组和pEGFP-N1空质粒转染组和对照组(P<0.01)。结论IDO基因表达的上调可能是诱导哮喘免疫耐受的机制之一。

Objective To observe the effects of mouse indoleamine 2,3-dioxygenase （IDO） gene on CD4^＋ T cells by transfecting an eukaryotic expression vector containing mouse IDO gene fused with enhanced green fluorescent protein （pEGFP-N1）. Methods The immature dendritic cells （imDCs） derived from mouse bone marrow were cultured in vitro and morphologically identified by transmission electron microscope, scanning electron microscope and flow cytometry. The immature dendritic cells were transfected by DOTAP liposome, and the expression of green fluorescence protein was confirmed with inverted fluorescence microscope. The spleen-derived CD4＋ T lymphocytes from mice were isolated and purified in vitro, and then carried to mixed lymphocyte reaction （MLR） with 3H-TdR incorporation. The effect of IDO gene on the proliferation of spleen-derived CD4＋ T lymphocytes of the allergized mouse challenged by ovalbumin was assayed by 3H-TdR MLR incorporation, and the apoptosis of spleen-derived CD4＋ T lymphocytes was assayed by TUNEL. Results The proliferation of spleen-derived CD4＋ T lymphocytes of the allergized mouse challenged by ovalbumin were inhibited by the imDCs transfected with IDO gene, the number of CD4＋ T lymphocytes treated with the imDCs transfected pEGFP-N1-IDO eukaryotic expression vector decreased significantly compared with that in control group （P〈0.01）. Apoptosis of the CD4＋ T lymphocytes was induced by the imDCs transfected with IDO gene, and the apoptotic rate in pEGFP-N1-IDO transfection group was 15.3%±2.6%, which was significantly higher compared with that of control group and the imDCs transfected pEGFP-N1 eukaryotic expression vector group （P〈0.01）. Conclusion The up-regulation of IDO gene expression may induce immunotolerance in asthma.