结直肠癌患者外周血 LAP ＋CD4＋调节性T 细胞的分离纯化及功能鉴定

摘要

Objective To explore a method for isolating and purifying LAP +CD4 +regulatory T (LAP +CD4 +T)cells from human peripheral blood of patients with colorectal carcinoma (CRC)by magnetic activated cell sorting (MACS)and to investigate the function of these T cells in vitro.Methods LAP +CD4 +T cells were isolated by MACS two-step method (negative sorting and positive sorting).The purity and the survival of these T cells were identified by flow cytometry (FACS)and Typan blue staining.The inhibition effect of LAP +CD4 +T cells on the proliferation of LAP -CD4 +T cells was determined by CCK-8.The expression of IL-10 and TGF-βwas measured by ELISA.Results The purity of LAP +CD4 +T cells isolated by MACS was 95.16% ±2.11%.The cell viability was 93.64% ±1.33%.The proliferation of LAP -CD4 +T was obviously inhibited by intervention of LAP +CD4 +T cells.The suppressor cytokine,TGF-βand IL-10,which were secreted by LAP +CD4 +T cells was more than that of LAP -CD4 +T cells.Conclusion MACS used for isolating LAP +CD4 +T cells from peripheral blood of patients withe CRC has advantages of high efficiency,high speed,and high purity and no damage to cell activity.%目的：应用免疫磁珠分选法分离纯化结直肠癌患者外周血中 LAP ＋CD4＋调节性 T 细胞（LAP ＋CD4＋T），并对分选获得的目的细胞进行体外功能的初步研究。方法按照免疫磁珠两步法（阴性分选和阳性分选）分离结直肠癌患者外周血单个核细胞中的 LAP ＋CD4＋T 细胞，流式细胞术（FACS）检测分选后细胞的纯度；台盼蓝染色检测细胞存活率；CCK-8掺入法检测其对 LAP －CD4＋T 细胞的增殖抑制效应；ELISA 法检测体外细胞因子 TGF-β和IL-10表达。结果经免疫磁珠法分选获得的 LAP ＋CD4＋T 细胞纯度为（95．16±2．11）％，台盼蓝染色细胞存活率为（93．64±1．33）％。CCK-8掺入法检测结果显示 LAP ＋CD4＋T 细胞干预后 LAP －CD4＋T 细胞生长明显受到抑制（P ＝0．000）；ELISA 结果显示 LAP ＋CD4＋T 细胞较 LAP －CD4＋T 细胞分泌更高浓度的细胞抑制因子 TGF-β和 IL-10（P ＝0．000）。结论免疫磁珠法分选结直肠患者外周血中 LAP ＋CD4＋T 细胞具有高效、快捷、纯度高等优点，且不损伤细胞活性。