分离外周血单个核细胞的条件优化  

Optimization of condition on peripheral blood mononuclear cells separation

作　　者：蔡敏敏 顾晓琼 高飞 邱先桃 陈小娟 刘非 郑浩 

机构地区：[1]广州市妇女儿童医疗中心检验科,广东广州510623

出　　处：《国际检验医学杂志》2016年第1期1-2,5,共3页International Journal of Laboratory Medicine

基　　金：广东省自然科学基金资助项目(S2013010012520);广州市科技计划资助项目(201510010159)

摘　　要：目的比较不同抗凝剂、离心力及离心时间对葡聚糖-泛影葡胺(Ficoll)密度梯度离心法分离单个核细胞的影响,优化实验方案。方法取肝素、EDTA及2种抗凝剂混合后的3种不同抗凝血,采用Ficoll法进行分离,用血细胞分析仪进行分类和计数,计算回收率和纯度;同时对EDTA抗凝血进行不同离心力及离心时间的处理,优化分离效果。结果 EDTA组获得PBMC回收率为(51.32±50.21)%,纯度为(45.38±22.14)%;肝素组获得PBMC回收率为(24.52±19.10)%,纯度为(78.46±11.91)%;EDTA+肝素组获得PBMC回收率为(56.32±24.30)%,纯度为(80.27±12.44)%。经单因素方差分析,PBMC回收率方面,EDTA组与肝素组比较差异有统计学意义(P<0.05),EDTA组回收率较高,而肝素+EDTA组与EDTA组回收率比较,差异无统计学意义(P>0.05);PBMC纯度方面,EDTA组与肝素组比较差异有统计学意义(P<0.05),肝素组PBMC纯度较高,而肝素+EDTA组与肝素组比较,纯度则差异无统计学意义(P>0.05);PBMC存活率方面,肝素+EDTA组与肝素组、EDTA组比较,均差异无统计学意义(P>0.05)。对EDTA抗凝血进行单个核细胞分离实验表明,在离心力为600×g(1 800r/min),离心时间为25min时分离效果最佳。结论肝素+EDTA组回收率、纯度与单种抗凝剂实验组相比,结果差异无统计学意义(P>0.05),故在实验过程中可以混合两种抗凝全血进行细胞分离。EDTA抗凝血在离心力为600g(1 800r/min)、离心时间为25min时,分离单个核细胞效果最佳。Objective To compare the influence of different anticoagulants,centrifugal force and the centrifugal time on separation of peripheral blood mononuclear cells（PBMC）by the Ficoll density gradient centrifugation method,and to optimize the experimental scheme.Methods Three different types of anticoagulation blood with heparin,EDTA and their mixture were separated by the Ficoll method.Then the sort and count were performed by using the blood cell analyzer.The recovery rate and purity were calculated;Meanwhile EDTA anticoagulation blood was treated by different centrifugal force and centrifugal time for optimizing the separation effect.Results The recovery rate and purity of PBMC in the EDTA group was（51.32±50.21）% and（45.38±22.14）%;which in the heparin group were（24.52±19,10）%and（78.46±11.91）%;which in the EDTA plus heparin group were（56.32＋24.30）% and（80.27＋12.44）% respectively.The single factor variance analysis showed that the recovery rate of PBMC had statistically significant difference between the EDTA group and the heparin group（P〈0.05）,the EDTA group had higher recovery rate,but the recovery rate had no statistically significant difference between the heparin plus EDTA group and the heparin group（P〉0.05）;in the aspect of the purity of PBMC,there was statistical difference between the EDTA group and heparin group（P〈0.05）,the heparin group had higher PBMC purity,but the purity had no statistical difference between the heparin group and the heparin plus EDTA group（P〈0.05）;in the aspect of the survival rate of PBMC,there was no statistically significantly differences between the heparin group with the heparin plus EDTA group and the heparin group with the EDTA group（P〉0.05）.The mononuclear cell separation experiments in the EDTA anticoagulation blood showed that in the centrifugal force 600×g（1 800r/min）and centrifugal time 25 min,the separation effect was optimal.Conclusion The recovery rate and purity of PBMC in the heparin plus EDTA group ha

关 键 词：单个核细胞 Ficoll连续密度梯度离心法 抗凝剂 分离效果 

分 类 号：R446.11[医药卫生—诊断学]