Foxp3转染对哮喘小鼠脾淋巴细胞功能的影响

Effects of Foxp3 transfection on splenocyte functions in asthma mice

目的:转染Foxp3至哮喘小鼠脾淋巴细胞,探讨Foxp3表达对脾淋巴细胞功能的影响。方法:卵白蛋白(OVA)致敏激发制作哮喘小鼠模型,收集培养脾脏淋巴细胞;使用电穿孔法转染真核表达载体pcDNA3.1(-)-Foxp3至脾脏淋巴细胞,并设转染空质粒组和对照组;RT-PCR和Western blotting检测Foxp3的表达;流式细胞术检测转染后CD4^+CD25^+Treg细胞/CD4^+细胞比例;MTT法检测转染后的脾脏淋巴细胞增殖反应,ELISA检测脾淋巴细胞上清中白细胞介素4(IL-4)和干扰素γ(IFN-γ)的含量。结果:转染组Foxp3 mRNA和蛋白的表达水平显著高于空质粒组和对照组;转染Foxp3后CD4^+CD25^+Freg细胞/CD4^+细胞比例显著高于空质粒组和对照组;与空质粒组和对照组相比,转染pcDNA3.1(-)-Foxp3质粒明显抑制了脾淋巴细胞增殖;转染组细胞上清中IL-4和IFN-γ含量低于空质粒组和对照组。结论:转染pcDNA3.1(-)-Foxp3至哮喘小鼠脾淋巴细胞,Foxp3得到有效表达。Foxp3的高表达能增加CD4^+CD25^+T细胞的数量,抑制哮喘小鼠脾淋巴细胞的增殖以及Th1和Th2细胞因子的产生。

AIM：To study the expression and the effects of Foxp3 on the immunologic functions by transfecting the Foxp3 eukaryotic expression plasmid into the splenocytes of the asthma mice.METHODS：The mice were sensitized and challenged by ovalbumin to make asthma model.The splenocytes were harvested and cultured.The Foxp3 expression vector pcDNA3.1（- ） -Foxp3 was transfected into the splenocytes with electroporation.The splenocytes transfected with empty vector[pcDNA3.1（- ）]and control splenocytes（non-transfected） were also set up.The expression of Foxp3 at mRNA and protein levels was detected by RT-PCR and Western blotting,respectively.The proportion of CD4^＋ CD25^＋ Treg cells/CD4^＋ cells was measured by flow cytometry.Proliferation of the splenocytes was analyzed with MTT assay. ELISA was used to determine the levels of interleukin 4（IL-4 ） and interferon -γ（IFN -γ） in the supernatant of the splenocytes.RESULTS;The expression of Foxp3 at mRNA and protein levels in transfection group was significantly higher than that in empty vector group and control group.The proportion of CD4^＋ CD25^＋ Treg cells/CD4 ＋ cells in transfection group was higher than that in empty vector group and control group.The proliferation of transfected cells was markedly inhibited compared with empty vector group and control group.The levels of IL-4 and IFN-y were significantly lower in transfection group than those in empty vector group and control group.CONCLUSION：The transfected Foxp3 gene overexpresses in the splenocytes of asthma mice.Foxp3 increases the number of CD4^＋ CD25^＋ T cells and inhibits the proliferation and production of Thl/Th2 cytokines in splenocytes.