Chin J Schisto Control ›› 2007, Vol. 19 ›› Issue (3): 192-195.

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

 Zhang Su-hua, Lin Dan-dan, Zhang Mei-juan, Chen Xue-song, Xu Jin-mei, Shan Hao, Wu Guan-ling, Wu Hai-wei   

  • Online:2013-01-15 Published:2013-01-21

Ficoll密度梯度离心法分离猪外周血单个核细胞条件的探讨

张素华|林丹丹|张美娟|陈雪松|徐进梅|单昊|吴观陵|吴海玮   

  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)
  • 作者简介:张素华(1981-)|女|硕士研究生。研究方向:血吸虫感染的分子免疫学

Abstract:

 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 ob-
served 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:l.llo ng/L ) by first centrifuging the samples at
1 500 r/ min (radius:15 cm) for 30 minutes at room temperature(20~25 'C), followed by washing
the cell pellets twice with D-Hank's medium at 1 500 r/min for 10 minutes at 4 'C. 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.

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(1 500 r/min,30 min)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1.110 ng/L),在20~25℃室温下以1 500 r/min(半径15 cm)离心30 min,接着低温(4℃)1 500 r/min离心10 min,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加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。

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

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