Ficoll密度梯度离心法分离猪外周血单个核细胞条件的探讨

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。

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

 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.