调节性CD4~+CD25~+T细胞的分离纯化及免疫功能研究

目的 制备调节性CD~+ CD25~+T细胞(Treg)分析其免疫功能,诱导局部免疫耐受防治同种异体复合组织移植(CTA)排斥反应.方法 采用免疫磁珠法(MACS)从雄性大鼠脾脏细胞分离CD4~+CD25~+Treg(1×10~6),2%锥虫蓝染色检测活性、流式细胞术分析其纯度,在5 mg/L抗CD3的刺激下观察其反应性、增殖及其与200 U/ml细胞介素(IL)-2的关系.结果 从8只雄性大鼠脾脏分选出的CD4~+CD25~+Treg活性平均为(97.90±0.36)%及纯度为(96.05±0.41)%,CD3刺激呈低反应,按比例培养抑制率为89%,IL-2可使CD4~+CD25~-抑制逆转.结论 MACS能快速分选出较高纯度的CD4~+CD25~+Treg,并且活性良好在体外具有免疫无能及免疫抑制作用,能满足动物CTA排斥反应研究的需要.

Isolation purification and function analysis of CD4~+CD25~+regulatory T cells

Objective To prepare CD4~+CD25~+regulatory T cells (Treg) and analyze their immune function in order to prevent and treat rejection of composite tissue allotransplantation (CTA) by inducing local immunotolence. Methods The CD4~+CD25~ +Treg (1×10~6) were isolated from the rat spleens,and sorted with magnetic bead by magnetic activated cell sorting (MACS) system. The activity of CD4~+CD25~+Treg was assayed by Trypan blue (2% ) staining,and their purity was analyzed by flow cytometry (FCM). The relationship between IL-2 (200 U/ml) and CD4~+CD25~+Treg reactivity to stimuli by CD3 (5 mg/L) antibody was observed. Results The mean activity and purity of CD4~+CD25~+Treg isolated from 8 male rats were (97.90±0.36)% and (96.05±0.41)% respectively. The lower reactivity to CD3 stimuli was found with cultivation inhibition ratio of 89%. IL-2 could reverse the inhibition. Conclusion The CD4~+CD25~+Treg with higher purity can be isolated quickly by MACS, and the cells are anergic immuno-suppression in vitro. This can fit the demand of investigation on animal CTA rejection.