大鼠CD4+CD25-T细胞的Foxp3基因转染及其表达***◆

背景：调节性T细胞在维持机体免疫应答稳态和免疫耐受方面具有非常重要的作用,但外周血中CD4+CD25+调节性T细胞含量极低,且增殖能力较差。 目的：以携带Foxp3基因的慢病毒EGFP+载体转染大鼠CD4+CD25- T细胞,观察其在大鼠CD4+CD25- T细胞中的表达。 方法：以免疫磁珠两步法分选大鼠CD4+CD25- T细胞,用携带大鼠Foxp3基因的慢病毒载体体外转染分选的细胞,以转染Foxp3基因的CD4+CD25- T细胞为实验组,EGFP空白质粒组及CD4+CD25- T细胞为阴性对照组,CD4+CD25+ T细胞为阳性对照组。荧光显微镜和RT-PCR分别从蛋白和mRNA水平检测Foxp3基因的表达。 结果与结论：成功完成了免疫磁珠的分选,获得了纯度较高的CD4+CD25-T细胞和CD4+CD25+ T细胞,细胞存活率为(94±2)%,慢病毒转染的CD4+CD25- T细胞高表达Foxp3基因。表明以携带Foxp3基因的慢病毒载体系统可有效介导Foxp3基因在大鼠CD4+CD25- T细胞中高表达。

Transfection of rat CD4+CD25- T cells with lentiviral vector containing Foxp3 gene and its expression

BACKGROUND: Regulatory T cells play an important role in maintaining immune response homeostasis and immune tolerance, but the CD4+CD25+T cells are less in peripheral blood and with low proliferation ability in vitro. OBJECTIVE: To observe the expression of transfected rat Foxp3 gene in rat CD4+CD25- T cells using lentiviral vector carrier EGFP+. METHODS: Rat CD4+CD25-T cells were sorted by magnetic activated cell sorting, and transfected by lentiviral vector containing rat Foxp3 gene in vitro. The whole observation was divided into three groups. Transfected rat CD4+CD25- T cells as the experimental group, the blank plasmid EGFP+ and rat CD4+CD25-T cells as the negative control group, rat CD4+CD25+T cells as the positive control group. The expression of Foxp3 gene was assayed by fluorescence microscope and RT-PCR from the level of protein and mRNA respectively. RESULTS AND CONCLUSION: The high purity CD4+CD25- and CD4+CD25+ T cells were obtained and the survival rate of cells was (94±2)%. Significantly high expression of rat Foxp3 gene was detected in lentiviral vector transfected CD4+CD25- T cells. The lentiviral vector system carrying rat Foxp3 gene can efficiently mediate the highly expression of rat Foxp3 gene in the rat CD4+CD25- T cells.