Percoll离心结合免疫磁珠分选的方法从外周血分离单核细胞  

Isolation of human peripheral blood monocytes using Percoll and immunomagnetic separation

作　　者：肖剑梅 何韦韦 王昊亮 万星 邓国宏 XIAO Jianmei;HE Weiwei;WANG Haoliang;WAN Xing;DENG Guohong(School of Life Sciences,Southwvest University,Chongqing 400715,China;Department of Infectious Diseases,Southvestern Hospital,Army Medical Universiry,Chongqing 400038,China)

机构地区：[1]西南大学生命科学学院,重庆400715 [2]陆军军医大学西南医院感染病科感染病研究重庆市重点实验室,重庆400038

出　　处：《免疫学杂志》2021年第5期454-460,共7页Immunological Journal

基　　金：国家自然科学基金重点项目(81930061);重庆市自然科学基金重点项目(cstc2019jcyj-zdxmX0004)。

摘　　要：目的探讨采用Percoll密度梯度离心结合免疫磁珠分选从人外周血富集白细胞层中分离提纯单核细胞的方法。方法采用Ficoll密度梯度离心分离淋巴细胞得到外周血单个核细胞,Percoll密度梯度离心从外周血单个核细胞中富集单核细胞,免疫磁珠阴性分选纯化单核细胞。流式细胞术检测细胞CD14和CD16的表达,分析单核细胞分离纯度。M-CSF诱导单核细胞分化为巨噬细胞。IFN-γ诱导巨噬细胞促炎性分化后,流式细胞术分析共刺激分子及HLA-DR的表达,RT-qPCR检测其细胞因子表达。结果Percoll分离结合免疫磁珠阴性分选分离出纯度为93.1%±1.5%的单核细胞。分离出的单核细胞呈煎蛋形,能成功分化为体积大、黏附力强、且呈纺锤状的巨噬细胞。与对照组相比,巨噬细胞在IFN-γ刺激下促炎性分化后,共刺激分子及HLA-DR表达显著上调,TNF-α、IL-6、CXCL10和CXCL11 mRNA表达也均显著上调。结论Percoll密度梯度离心结合免疫磁珠阴性分选能分离出高纯度且具有功能活性的单核细胞。Monocytes/macrophages play key roles in the pathogenesis of many immune-related diseases.Monocytes of high purity and functionality are required for the in-depth study of this cell population.In this study,we developed a method of human monocytes separation with the combination of Percoll density gradient centrifugation and immunomagnetic separation.The peripheral blood mononuclear cells were isolated from human peripheral blood by Ficoll density gradient centrifugation.Monocytes were firstly enriched from peripheral blood mononuclear cells using Percoll density gradient centrifugation,and then purified by immunomagnetic beads negative separation.The purity of the isolated monocytes was evaluated by determining CD14 and CD16 expression using flow cytometry.Then,M-CSF was used to induce the differentiation of monocytes into macrophages.After the macrophages pro-inflammatory differentiation induced by IFN-γ,the expression of co-stimulatory molecules and HLA-DR were analyzed by flow cytometry and the mRNA of pro-inflammatory cytokines were evaluated by RTqPCR.Data showed that the purity of the isolated monocytes was 93.1%±1.5%.M-CSF could promote the differentiation of fried egg shaped monocytes into spindle shaped macrophages with large volume and strong adhesion.IFN-γstimulation induced successful pro-inflammatory differentiation of cultured macrophage,as revealed by up-regulated costimulating molecules HLA-DR,TNF-α,IL-6,CXCL10 and CXCL1.In conclusion,the isolation method is constructed with property of simple,inexpensive and time-effective,and the isolated monocytes demonstrate high purity and functionality.

关 键 词：PERCOLL 免疫磁珠分选 单核细胞 巨噬细胞 分离 

分 类 号：R392-33[医药卫生—免疫学]