【摘要】 目的探索利用磁性细胞分离(MACS)系统高效、快速地分离大鼠脾脏CD4+CD25+调节性T细胞。方法采用免疫磁珠两步法分离大鼠脾脏内的CD4+CD25+调节性T细胞,首先采用“鸡尾酒”抗体和抗IgG磁珠阴性分选CD4+T细胞,再用抗CD25PE抗体和抗PE磁珠阳性分选获得CD4+CD25+T细胞。分离后的细胞经流式细胞仪检测分离纯度;台盼蓝染色检测细胞存活率;体外增殖实验检测其对CD4+CD25-T细胞的免疫抑制作用。结果阴性分选获得的CD4+T细胞纯度为(83.6±2.5)%(79%～87%),阳性分选后获得的CD4+CD25+T细胞纯度为(90.2±1.8)%(86%～93%),细胞存活率为(92.8±3.4)%(92%～95%),体外增殖实验表明,CD4+CD25+T细胞能明显抑制CD4+CD25-T细胞的增殖(P<0.01)。结论采用MACS系统阴性加阳性分选可以高效、快速地获得理想纯度和有免疫抑制功能的大鼠CD4+CD25+调节性T细胞。更多还原

【Abstract】 Objective To find a stable and efficient method for ex vivo concentration of CD4+CD25+ regulatory T cells in rats. Methods CD4+CD25+ regulatory T cells were separated from the rat splenic cells in two steps by magnetic cell sorting (MACS) system. CD4+ T cells were first negatively sorted by cocktail antibodies and anti-IgG magnetic microbeads. And then CD4+CD 25+T cells were positively sorted by anti-CD25 PE and anti-PE magnetic microbeads. The purity and the cell survival rate of the sorted cells were measured by flow cytometry and trypan blue dyeing respectively, and the immunosuppressive action of CD4+CD25+ T cells on the proliferation of CD4+CD25- T cells was also assessed by in vitro cell proliferation assay. Results The purity of negatively sorted CD4+ T cells were (83.6±2.5)% (79%~87%), and the purity of positively sorted CD4+CD25+ T cells was (90.2±1.8)% (86%~93%) with the survival rate of (92.8±3.4)% (92%~95%). These concentrated cells significantly suppressed the proliferation of CD4+CD25- T cells in mixed lymphocyte culture (CD25+/CD25- versus CD25-, P<0.01). Conclusion We created a two-step procedure of magnetic cell sorting system for CD4+CD25+ regulatory T cells sorting, which insures the cells to be satisfactorily purified and well functioned.更多还原