小鼠CD4^+CD25^+T细胞的分离、鉴定和体外功能的检测  

Isolation, identification, and function of mouse CD4^+CD25^+ regulatory T cells

作　　者：周洁[1] 袁劲[1] 周鸿敏[1] 杜敦峰[1] 陈必成[1] 昌盛[1] 陈忠华 

机构地区：[1]华中科技大学同济医学院附属同济医院同济器官移植研究院器官移植教育部卫生部重点实验室,武汉430030

出　　处：《免疫学杂志》2005年第2期92-95,99,共5页Immunological Journal

基　　金：国家 973资助项目 (2 0 0 3CB5 15 5 0 5 )

摘　　要：目的　建立CD4 + CD2 5 + T细胞的免疫磁性分离(MACS)方法,并检测其体外免疫抑制功能。方法　分离C5 7BL 6小鼠和Balb c小鼠脾脏细胞后运用MACS法分离CD4 + CD2 5 + T淋巴细胞,用台盼蓝染色法和流式细胞法检测选出细胞的活性和纯度,用淋巴细胞转化实验分析其免疫抑制功能。结果　MACS分选法分选出的CD4 + CD2 5 + T细胞活性>97% ,纯度>95 % ;此方法获得的CD4 + CD2 5 + T细胞在体外不增殖,经丝裂原活化后可以抑制同品系小鼠和异品系小鼠的CD4 + CD2 5 -T细胞增殖,这种抑制作用具有剂量依赖性。结论　MACS法可简单迅速分选出高纯度无菌CD4 + CD2 5 + T细胞,且不影响细胞活力。CD4 + CD2 5 + T调节性细胞在体外具有免疫无能和免疫抑制作用;丝裂原活化的该细胞免疫抑制功能具有剂量依赖性,但不具有抗原特异性。Objective To establish a method for isolating CD4 +CD25 + T cells, and to identify the purity and the function of these cells in vitro. Methods After the splenocytes of C57BL/6 and Balb/c mice were separated, the CD4 +CD25 +T cells were isolated by magne- tic activated cell sorting (MACS). The activity and the purity of the isolated cells were detected by trypan blue staining and fluorescence-activa- ted cell sorting (FACS), respectively. The immunosuppression effects of the isolated cells effect on the CD4 + T cells proliferation was analyzed by proliferation assays. Results The activity of the CD4 +CD25 +T cells was beyond 97%, and the purity of them was over 95%. In co-culture experiments, the CD4 +CD25 + T cells could suppress the activation and proliferation of CD4 + T cells. The mitogen-activated CD4 +CD25 + regulatory T cells from the mouse of the same or different stain can inhibit the proliferation of CD4 +CD25 -T cells in manner of dose- dependence. Conclusion MACS isolation kit can isolate CD4 +CD25 +T cells effectively with on effects on their activation. The CD4 +CD25 + T cells have the characteristics of immune anergy and immune inhibition. In a conclusion, the immune inhibition of activated CD4 +CD25 + regulatory T cells doesn't have antigen specificity in vitro, but the intensity of the inhibition dependents on the dosage.

关 键 词：CD4^+CD25^+T细胞 免疫磁性分离 AlamarBlue 免疫无能 免疫抑制 

分 类 号：R392[医药卫生—免疫学]