恒河猴外周血调节性T淋巴细胞的分离纯化及鉴定

目的建立恒河猴外周血CD4+CD25+调节性T淋巴细胞(regulatory T cells,Tregs)快速有效的分离纯化方法。方法 4～5岁健康恒河猴10只,雌性4只,雄性6只,体重5～8 kg;于大隐静脉抽取外周血,每只抽取8 mL。密度梯度离心法分离外周血单个核细胞(peripheral blood mononuclear cell,PBMC),分别采用非人灵长类Tregs分离试剂盒的生物素标记的混合抗体和磁珠标记的抗生物素抗体阴性分选,以及大鼠抗人CD4-活化蛋白C(activatedprotein C,APC)和抗APC多选试剂盒中的磁珠标记的抗APC抗体阳性分选出CD4+T淋巴细胞,比较两种方法获得细胞的得率、活性及纯度;选择得率、活性和纯度较高的分选方法获得的CD4+T淋巴细胞,用磁珠标记的抗人CD25抗体进行阳性分选,获得CD4+CD25+Tregs,流式细胞仪检测纯度、活性及FoxP3表达水平,以及Tregs对刀豆蛋白(concanavalinA,ConA)刺激的自体CD4+CD25-效应性T淋巴细胞(effective T cells,Teffs)增殖的抑制功能。结果 CD4+T淋巴细胞阳性分选和阴性分选后,细胞活性均达95%左右,差异无统计学意义(P>0.05),但阳性分选后CD4+T淋巴细胞得率和纯度均显著高于阴性分选(P<0.05)。后续CD4+CD25+Tregs分选采用阳性分选法获得的CD4+T淋巴细胞。经双阳性分选收集的目的细胞中CD4+CD25+Tregs占76.2%±8.6%,活细胞比例为93.3%±4.7%,FoxP3阳性细胞比例为74.2%±6.9%。混合培养后Tregs均对ConA刺激的Teffs的增殖有抑制作用。结论免疫磁珠双阳性分选法能有效分选出恒河猴外周血中有功能的CD4+CD25+Tregs。

ISOLATION AND IDENTIFICATION OF REGULATORY T CELLS IN PERIPHERAL BLOOD OF RHESUS MONKEYS

Objective To establish a method to isolate the CD4+CD25+ regulatory T cells(Tregs) and to identify the purity and function of these cells.Methods The peripheral blood(8 mL) were collected from the great sflaphenous vein of 10 rhesus monkeys(4 females and 6 males,aged 4-5 years,and weighing 5-8 kg).The mononuclear cells were isolated with density gradient centrifugation.CD4+T cells were separated by the Magnetic cell sorting(MACS) negative selection and MACS positive selection.The cell yield rate,the cell viability,and the cell purity were compared between MACS negative selection and MACS positive selection.In CD4+ MACS negative selection,the anti-biotin MicroBeads and biotin-antibody cocktai in CD4+CD25+Tregs isolation kit non-human primate were used,and in MACS positive selection,the anti-APC MicroBeads in CD4+CD25+Tregs isolation kit non-human primate and CD4-APC were used.The CD4+T cells separated by positive selection were selected to obtain CD4+CD25 Tregs with CD25 MicroBeads.The purity,activity,the FoxP3 level,and the suppressive function to concanavalin A(ConA) activated autologous CD4+CD24-effective T cells(Te s) of CD4+CD25+Tregs were detected by ow cytometry.Results After CD4+T cells were separated by MACS negative selection and MACS positive selection,the cell viabilities were all up to 95%,showing no signi cant difference(P > 0.05).The cell yield rate and purity of CD4+T cells by positive selection were signi cantly higher than those of CD4+T cells by negative selection(P < 0.05).CD4+CD25+Tregs can be successfully isolated by MACS double positive selection.The classifying purity was 76.2% ± 8.6%;the cell activity was 93.3% ± 4.7%;and the level of FoxP3 was 74.2% ± 6.9%.The CD4+CD25+Tregs had suppressive effect on ConA activated autologous CD4+CD25-Te s.Conclusion MACS double positive selection can be used to isolate high-purity CD4+CD25+Tregs from the peripheral blood of rhesus monkeys and the process does not in uence the activity of CD4+CD25+Tregs.