A simple method for removing low-density granulocytes to purify T lymphocytes from peripheral blood mononuclear cells
A simple method for removing low-density granulocytes to purify T lymphocytes from peripheral blood mononuclear cells
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一种简单的去除低密度粒细胞从外周血单个核细胞中纯化T淋巴细胞的方法
DOI：
10.1631/jzus.b1600064
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发表时间：
2017-07-01
影响因子：
5.1
通讯作者：
Wang, Guo-chun
中科院分区：
文献类型：
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作者：
Zhang, Si-gong;Song, Yu-xin;Wang, Guo-chun
低密度粒细胞（LDG）可以自发地和过度地形成中性粒细胞胞外陷阱（NET）。当用外周血单个核细胞（PBMC）研究T淋巴细胞时，PBMC中含有的LDG可通过形成NET降低T淋巴细胞活化的阈值。本研究主要探讨常见自身免疫性疾病中LDGs的表达谱及从PBMCs中去除LDGs的方法。采用流式细胞术检测55例皮肌炎（DM）、15例多发性肌炎（PM）、42例类风湿性关节炎（RA）、25例系统性红斑狼疮（SLE）患者和19例健康对照者外周血单个核细胞（PBMC）中LDG的含量。探讨并比较了三种去除LDG的方法。取出后，再次检测6例T-SPOT.TB阳性患者的PBMC，以了解PBMC中含有的LDG是否会影响T淋巴细胞反应。DM患者的PBMCs中LDG百分比显着较高（8.41±10.87）%，P<0.0001），PM RA组（4.05±6.97）%，P = 0.0249），SLE组（7.53±11.52）%，P =0.0006）与对照组（1.28±0.73）%比较差异有统计学意义（P <0.0001）。去除LDG后，T-SPOT.TB值显著降低。在一定范围内增加相对离心力（RCF）可使LDG百分比从初始的高水平下降，但不能显著增加LDG清除率。与全血沉淀法相比，PBMC贴壁法能显著去除LDG，而对PBMC中T淋巴细胞百分率影响不大。SLE、DM、PM和RA患者PBMC中LDG百分比显著增加。LDGs对PBMC培养中T淋巴细胞的影响不容忽视。贴壁法是一种简单易用的从PBMC中去除LDG和纯化T淋巴细胞的方法。
Low-density granulocytes (LDGs) can form neutrophil extracellular traps (NETs) spontaneously and excessively. When peripheral blood mononuclear cells (PBMCs) are used for studying T lymphocytes, LDGs contained in the PBMCs may decrease the threshold of activating T lymphocytes by forming NETs. This study focused on the profiles of LDGs in common autoimmune diseases and methods for removing LDGs from PBMCs. The percentages of LDGs in PBMCs from 55 patients with dermatomyositis (DM), 15 with polymyositis (PM), 42 with rheumatoid arthritis (RA), 25 with systemic lupus erythematosus (SLE), and 19 healthy controls were determined by flow cytometry. Three methods of removing LDGs were explored and compared. After removal, PBMCs from six patients with positive T-SPOT.TB were tested again to find out if LDGs contained in the PBMCs could influence T lymphocyte reactions. Significantly higher LDG percentages were found in PBMCs from patients with DM ((8.41±10.87)%, P<0.0001), PM ((8.41±10.39)%, P<0.0001), RA ((4.05±6.97)%, P=0.0249), and SLE ((7.53±11.52)%, P=0.0006), compared with the controls ((1.28±0.73)%). The T-SPOT.TB values significantly decreased after LDGs were removed. Increasing relative centrifugal force (RCF) within a limited range can decrease the LDG percentage from an initial high level, but not markedly increase the LDG clearance rate. Compared with the whole blood sediment method, the PBMC adherence method can significantly remove LDGs yet scarcely influence the T lymphocyte percentage in PBMCs. The LDG percentage in PBMCs is significantly increased in patients with SLE, DM, PM, and RA. The influence of LDGs on T lymphocytes cannot be ignored in PBMC cultures. The adherence method is a simple and easy-to-use method for removing LDGs and purifying T lymphocytes from PBMCs.