人外周血单核细胞的分离和电穿孔法转染

目的:建立分离纯化人外周血单核细胞及转染的有效方法。方法:应用基于HISTOPAQUE的密度梯度离心及抗体标记纯化的方法分离单核细胞,并以电穿孔技术转染绿色荧光蛋白(GFP)表达质粒、肿瘤坏死因子受体相关因子6(TRAF6)小干扰RNA。结果:基于外周血单核细胞的表面标志分子CD14的流式细胞分析表明得到了高纯度(89.95%)的外周血单核细胞;GFP荧光照片显示GFP表达质粒的转染效率为60%～70%;免疫印迹显示TRAF6的表达抑制(90%)下调了脂多糖(LPS)对JNK的激活,说明通过电穿孔技术有效递送了TRAF6小干扰RNA,表明单核细胞若缺少TRAF6将抑制LPS-TLR4信号通路的激活。结论:建立了一种高效的从人外周血中分离原代单核细胞的方法,且应用基于NucleoFectorⅡ的电穿孔技术实现了对此原代单核细胞的高效转染,为进一步外周血单核细胞的相关功能研究奠定了方法学基础。

Isolation of Human Monocytes from Peripheral Blood and Transfection via Electroporation

Objective： To establish an efficient method for isolating human peripheral monocytes from peripheral blood and for transfecting monocytes.Methods： A method based on density-gradient centrifugation and further magnetic beads conjugated with antibodies was used for isolating monocytes from human peripheral blood,and then the cells were transfected by a modified electroporation method with GFP-expressing vector or siRNA to knockdown the expression of TNF receptor associated factor 6（TRAF6）.Results： Monocytes with high purity were successfully isolated from peripheral blood（purity above 89.95%） based on FACS analysis using monocyte marker CD14.The electroporation method delivered plasmid DNA（transfection efficiency about 60%～70% based on GFP expression）,and also small interference RNA into monocytes efficiently by checking TRAF6 expression（knocking down efficiency above 90%） using Western blot method,and siRNA knocking down TRAF6 inhibits the activation of JNK by LPS treatment.Conclusion： The method reported here was very efficient for isolating human primary monocytes from peripheral blood,and the NucleoFectorⅡ-based electroporation was capable of transfecting the primary monocytes efficiently.Together,these would provide methodological basics for future functional studies based on monocytes.