不同抗凝剂对外周单个核细胞增殖影响比较、γδ T细胞鉴定

Influence of different anticoagulants on proliferation of peripheral mononuclear cells and identification for γδ T cell

目的探讨3种抗凝剂保存的外周血对培养γδT细胞的增殖和杀伤功能的影响研究。方法选择获得知情同意的6例健康志愿者,其中男性3例,女性3例;年龄25~45岁。常规无菌抽取新鲜外周血15 mL。将其外周血标本分别置于肝素钠、细胞保存液(枸橼酸钠)和乙二胺四乙酸(EDTA)抗凝剂中(即肝素钠组、细胞保存液组、EDTA组),并立即处理培养细胞,用CCK-8法检测3组细胞的增殖情况。在第10天,行流式细胞术检测3组γδT细胞的穿孔素、颗粒酶B和CD107a的表达。结果在外周血细胞培养过程中,EDTA组细胞增殖不明显;肝素钠组和细胞保存液组细胞增殖明显,在第14天细胞增殖倍数最大分别为137.00%±1.44%和99.00%±1.45%,且肝素钠组优于细胞保存液组(t=2.72,P<0.01)。流式细胞仪检测发现肝素钠组γδT细胞穿孔素、颗粒酶B和CD107a的表达都明显高于细胞保存液组(t=3.871,P=0.003;t=2.744,P=0.021;t=2.261,P=0.047)。结论肝素钠和细胞保存液均可用于γδT细胞培养的血液抗凝,但肝素钠保护γδT细胞的杀伤功能明显优于细胞保存液。

Objective To investigate the effects of three anticoagulants preserved peripheral blood samples on the proliferation and killing ability of cultured γδT cell. Methods Six healthy volunteers were enrolled, which included 3 males and 3 females, aged 25 - 45 years old. Fifteen mL peripheral blood was collected, heparin sodium（heparin sodium group）, cell preservation liquid（sodium citrate, cell preservation liquid group） and ethylene diaminete traacetic acid（EDTA group） were added respectively, and the proliferation of γδT cells was determined by CCK-8 assay after cultured. The expression of perforin, granzyme B and CD107a were detected by flow cytometry on the 10th day. Results The proliferation of γδTcell was not obvious in EDTA group, but obvious in heparin sodium group and cell preservation liquid group. The multiple cell proliferations on the 14th day were maximum（137.00 %± 1.44 % and 99.00 % ±1.45 %, respectively）, and the proliferation in heparin sodium group was better than that of cell preservation liquid group（t = 2.72, P 〈 0.01）. Higher expression of perforin, granzyme B and CD107a of γδT cell were observed in heparin sodium group than that of cell preservation liquid group（t = 3.871, P = 0.003; t = 2.744, P = 0.021; t = 2.261, P = 0.047）. Conclusion It is demonstrated that the heparin sodium and cell preservation liquid could be used in blood sample anticoagulation for γδT cell culture, while the killing ability of γδT cell by heparin sodium group is better than that of cell preservation liquid.