Ficoll密度梯度离心法分离猪外周血单个核细胞条件的探讨  

An exploration of the conditions for isolating peripheral blood mononuclear cells from pig's blood using Ficoll density gradient centrifugation

作　　者：张素华[1] 林丹丹[1] 张美娟[1] 陈雪松[1] 徐进梅[1] 单昊[1] 吴观陵[1] 吴海玮[1] 

机构地区：[1]南京医科大学病原生物学系,江苏省现代病原生物学重点实验室,南京210029

出　　处：《中国血吸虫病防治杂志》2007年第3期192-195,共4页Chinese Journal of Schistosomiasis Control

基　　金：国家自然科学基金重点项目(30430600)

摘　　要：目的探讨猪外周血单个核细胞(PBMC)最佳分离条件,为寄生虫免疫学研究提供有效的方法。方法使用猪淋巴细胞分离液,在不同离心转速和时间条件下分离猪PBMC,观察所得云雾状PBMC层的厚度,计算细胞得率及细胞活力,以确定猪淋巴细胞分离液分离猪PBMC的最佳实验条件。同时采用文献报道的人淋巴细胞分离液分离猪PBMC的分离条件(1500r/min,30min)分离猪PBMC,比较两者分离PBMC效果。结果使用猪淋巴细胞分离液(密度1.110ng/L),在20～25℃室温下以1500r/min(半径15cm)离心30min,接着低温(4℃)1500r/min离心10min,洗涤2次,这样获得的猪PBMC效果最好。同样条件下,用人的淋巴细胞分离液分离猪PBMC,不仅所得细胞沉淀中混杂细胞如红细胞及其他细胞碎片较多,而且PBMC细胞得率及活力也不好。结论本实验提出了获得大量有活力的猪PBMC的方法和条件。Objective To explore the best condition isolating the peripheral blood mononuclear cells（PBMC）from pig＇s blood in order to provide an effective method for parasite immunology. Methods PBMCs from pig＇s blood were isolated with pig＇s lymphocyte separation medium （LSM） at different rotation speeds for different periods, the thickness of cloudlike PBMC layer was observed and the yield of obtained PBMC and their activity were caculated. Simultaneously, PBMCs from pig＇s blood were isolated with human LSM under the conditions reported by previous papers （1 500 r/min,30 min） and the isolating efficiency was compared with that of pig＇s LSM. Results The best result was got with pig＇s LSM（density：1. 110 ng/L ） by first centrifuging the samples at 1 500 r/min （radius ：15 cm） for 30 minutes at room temperature （20-25℃ ）, followed by washing the cell pellets twice with D-Hank＇s medium at 1 500 r/min for 10 minutes at 4 ℃. PBMC that were isolated with human LSM from pig＇s blood under the same condition were impure and mixed with other cells such as red cells and other cell debris, and both the yield of obtained PBMC and their activity were low. Conclusion This study offered a method to get a large quantity of active PBMC.

关 键 词：猪 外周血单个核细胞(PBMC) Ficoll密度梯度离心法 寄生虫学 免疫学 

分 类 号：R53[医药卫生—内科学]