脂质体介导的IDO基因转染未成熟树突状细胞对其成熟性及功能的影响

The maturation characteristics and function of immature dendritic cells transfected by liposome-mediated indoleamine 2,3-dioxygenase(IDO)gene

目的:探讨脂质体介导的IDO基因转染对未成熟树突状细胞(Immature dendritic cells,imDCs)的成熟性及功能的影响。方法:从BALB/c小鼠骨髓培养imDCs,形态学观察后用流式细胞术鉴定imDC后,将其分为IDO+-imDC组:重组pEGFP-N1-IDO质粒在脂质体介导下转染imDCs;IDO--imDC组:pEGFP-N1空质粒转染imDCs;imDCs组:imDCs不做特殊处理;mDC组:将培养的imDC用TNF-α诱导成熟。流式细胞术检测IDO基因转染imDC细胞后细胞表型是否发生变化。Western blot检测各组IDO蛋白表达。混合淋巴细胞反应检测各组在体外刺激T淋巴细胞增殖的能力。结果:BALB/c小鼠骨髓培养的细胞表面标志和细胞形态符合imDCs典型特征;Western blot结果显示IDO+-imDC组可见IDO蛋白表达;IDO+-imDC组细胞表面分子CD80、CD86、MHCⅡ表达率分别为(9.4±2.2)%、(8.7±1.1)%、(11.4±2.6)%,imDC组分别为(8.5±1.8)%、(7.5±1.6)%、(10.2±2.1)%,两组比较无显著差异(P均>0.05),IDO+-imDC组在体外刺激T淋巴细胞增殖的能力明显低于imDC组(678±90.3vs1199±275.5,P<0.01)。结论:脂质体介导的IDO基因转染imDCs能维持细胞于未成熟状态,在MLR中,对T细胞增殖有明显的抑制作用。

Objective：To investigate the influence of indoleamine 2,3-dioxygenase（IDO）gene mediated by liposome transfection on function of immature dendritic cells（imDCs）.Methods：ImDCs were cultured from bone marrow of BALB/c mouse.The cells were identified by transmission electron microscope,scanning electron microscope,and flow cytometry.Then the imDCs were divided intoIDO＋-imDC group（imDCs transfected with recombinant pEGFP-N1-IDO expression vector）,IDO--imDC group（imDCs were transfected with recombinant pEGFP-N1 vector）,control group（imDCs without special processing）and mDC group（（imDCs induced with TNF-α）.IDO protein expression in each group was detected by Western blot;The expression of cell marker was determined with flow cytometry.Their ability to stimulate antigen-specificity T lymphocyte proliferation was determined with allogenetic mixed leukocyte reaction in each groups.Results：The cultured imDCs of BALB/c mouse bone marrow exhibited typial characteristics of surface markers and cell morphology consistent with imDCs;IDO protein expression was seen in transfection group.The expression rate of CD80,CD86,MHCⅡ was（9.4±2.2）%,（8.7±1.1）% and（11.4±2.6）%,respectively,which was exhibited no difference compared with those in imDC group（8.5±1.8）%,（7.5±1.6）% and（10.2±2.1）%,respectively,P0.01.The ability of IDO＋-imDC group to stimulate antigen-specificity T lymphocyte proliferation in vitro was distinctly lower than in imDC group（678±90.3 vs 1 199±275.5,P0.01）.Conclusion：ImDCs transfected by liposome-mediated IDO gene remain their immature state and can inhibit the proliferation of the T lymphocytes.