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目的:系统性红斑狼疮(SLE)患者外周血单个核细胞(PBMCs)中的低密度粒细胞(LDGs)可以自发而过度地形成中性粒细胞胞外网状陷阱(NETs),而形成的NETs会降低T细胞激活的阈值。采用PBMCs做淋巴细胞研究时,所含的异常增多的LDGs可能会显著影响实验结果。因此需要了解其他自身免疫病中LDGs的比例,并需要一种简便地祛除PBMCs中LDGs的方法。创新点:首次提出LDGs清除的方法,验证了离心后贴壁法清除LDGs的有效性和可靠性。方法:采用流式细胞仪测定55例皮肌炎(DM)患者、15例多发性肌炎(PM)患者、42例类风湿关节炎(RA)患者、25例SLE患者(阳性对照)和19例健康对照(阴性对照)PBMCs中LDGs的比例。采用提高离心力、离心后贴壁和全血沉淀后再离心这三种方法祛除PBMCs中的LDGs,进一步比较各种方法的LDGs清除率和对PBMCs中T细胞的影响程度。清除T-SPOT.TB阳性患者PBMCs中LDGs后复测T-SPOT.TB,明确LDGs是否对T-SPOT.TB结果有影响。结论:本研究中DM、PM、RA和SLE患者PBMCs中LDGs比例普遍升高(图1)。清除PBMCs中LDGs后,T-SPOT.TB显著降低(图2)。离心后贴壁法不但能最有效地清除LDGs,且对PBMCs中T细胞的比例影响最小(图3~5)。综上所述,PBMCs中LDGs的比例在DM、PM、RA和SLE患者中普遍增高;采用PBMCs做T细胞相关研究时LDGs通过形成NETs对T细胞激活的影响不能忽视;离心后贴壁法可以简单而高效地清除PBMCs中的LDGs。
OBJECTIVE: Low density neutrophils (LDGs) in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) spontaneously and overexpressed neutrophil extracellular mesh traps (NETs), while the formation of NETs Will lower the threshold for T-cell activation. The use of PBMCs in lymphocyte studies, the abnormal increase in LDGs may significantly affect the experimental results. There is therefore a need to understand the proportion of LDGs in other autoimmune diseases and to find a way to simply eliminate LDGs in PBMCs. Innovation: For the first time proposed LDGs removal method to verify the efficiency and reliability of the removal of LDGs by adherence method after centrifugation. Methods: Fifty-five patients with dermatomyositis (DM), 15 patients with polymyositis (PM), 42 patients with rheumatoid arthritis (RA), 25 patients with SLE (positive control) and 19 Example Healthy controls (negative control) Ratio of LDGs in PBMCs. Three methods were used to eliminate LDGs in PBMCs, such as increasing centrifugal force, adhering after centrifugation and re-centrifugation after whole blood sedimentation, and further comparing the clearance rates of LDGs and T lymphocytes in PBMCs. T-SPOT.TB was measured after LDGs in PBMCs of T-SPOT.TB positive patients were cleared, and whether LDGs affected the T-SPOT.TB results was determined. Conclusions: The proportion of LDGs in PBMCs among patients with DM, PM, RA and SLE is generally elevated in this study (Figure 1). T-SPOT.TB was significantly reduced after clearance of LDGs in PBMCs (Figure 2). After centrifugation, the adherent method not only cleared LDGs most effectively but also had the least effect on the proportion of T cells in PBMCs (Figures 3 to 5). In conclusion, the proportion of LDGs in PBMCs is generally increased in patients with DM, PM, RA and SLE. The effect of LDGs on the activation of T cells through the formation of NETs can not be ignored when PBMCs are used in T-cell related studies. Remove LDGs from PBMCs simply and efficiently.