吲哚胺2,3-双加氧酶基因转染未成熟树突状细胞对卵蛋白致敏小鼠模型CD_4^+T细胞的影响

Effects of indoleamine 2,3-dioxygenase gene-modified immature dendritic cells on spleen CD_4^+T cells in ovalbumin allergic 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试验,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,pEGFP-N1-IDO质粒转染组CD4+T细胞减少,与和pEGFP-N1空质粒转染组和对照组相比差异有统计学意义(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)%,较对照组和pEGFP-N1空质粒转染组显著增加(P<0.01)。结论IDO基因表达的上调可能成为诱导哮喘免疫耐受的作用和机制之一。

Objective To observe the effects of mouse IDO gene on CD4＋T cells by transfecting a eukaryotic expression vector containing mouse IDO gene fused with enhanced green fluorescent protein（pEGFP-N1）.Methods The immature dendritic cells derived from mouse bone marrow were cultured invitro and identified by morphology,transmission electron microscopy scanning electron microscopy and flow cytometry.The immature dendritic cells were transfected by DOTAP liposome and the green fluorescence protein expression was confirmed by inverted fluorescence microscopy.The spleen-derived CD4＋T lymphocytes from mice were isolated and pured invitro and then were carried through mixed lymphocyte reaction（MLR）.The effects of IDO gene on spleen-derived CD4＋T lymphocyte proliferation of the allergic mouse challenged by ovalbumin were assayed by 3H-TdR（MLR） incorporation and apoptosis of spleen-derived CD4＋T lymphocyte by TUNEL.Results The proliferation of spleen-derived CD4＋T lymphocyte of the allergic mouse challenged by ovalbumin was inhibited by imDCs transfected with IDO gene,the number of CD4＋T lymphocytes treated with imDCs transfected pEGFP-N1-IDO eukaryotic expression vector decreased significantly compared with that in the control group（P0.01）.The apoptosis of the CD4＋T lymphocyte was induced by the immature dendritic cells transfected by IDO gene,and the apoptosis rates were（15.3±2.6）%.Compared with imDCs transfected pEGFP-N1 eukaryotic expression vector and control,the difference was highly significant respectively（P0.01）.Conclusion The up-regulation of IDO gene expression may induce the immunotolerance in asthma.