Rodenburg R J, van den Hoogen F H, van de Putte L B, van Venrooij W J
Department of Biochemistry, University of Nijmegen, The Netherlands.
J Immunol Methods. 1998 Dec 1;221(1-2):169-75. doi: 10.1016/s0022-1759(98)00183-5.
We have compared three monocyte isolation procedures for their suitability in the analysis of cytokine mRNA expression in circulating monocytes. Monocytes were isolated from peripheral blood (1) using antiCD14 coated magnetic beads, (2) by Ficoll centrifugation, or (3) by Ficoll centrifugation followed by plastic adherence. The effect of the isolation procedure on the cytokine mRNA expression levels of the isolated monocytes was evaluated by RT-PCR. Results show that the expression of cytokine mRNAs determined in monocytes isolated using antiCD14 coated magnetized beads reflects best the cytokine mRNA levels in circulating monocytes. We subsequently applied this method to the analysis of cytokine mRNA expression levels in rheumatoid arthritis and control monocytes, which revealed that RA and control monocytes isolated by antiCD14 beads produce similar, very low TNF alpha, IL-1beta, and IL-8 mRNA levels.
我们比较了三种单核细胞分离方法在分析循环单核细胞中细胞因子mRNA表达方面的适用性。单核细胞从外周血中分离出来的方法有：（1）使用抗CD14包被的磁珠；（2）通过Ficoll离心法；或（3）先通过Ficoll离心法，然后进行塑料黏附法。通过逆转录聚合酶链反应（RT-PCR）评估分离方法对分离出的单核细胞中细胞因子mRNA表达水平的影响。结果表明，使用抗CD14包被的磁化磁珠分离的单核细胞中测定的细胞因子mRNA表达最能反映循环单核细胞中的细胞因子mRNA水平。我们随后将该方法应用于类风湿性关节炎和对照单核细胞中细胞因子mRNA表达水平的分析，结果显示，通过抗CD14磁珠分离的类风湿性关节炎和对照单核细胞产生相似的、非常低的肿瘤坏死因子α（TNFα）、白细胞介素-1β（IL-1β）和白细胞介素-8（IL-8）mRNA水平。
