一种经济高效的人外周血NK细胞纯化方法的建立

Establishment of an economical method to obtain NK cells with high purity

目的:建立一种经济并且纯化率高的人外周血NK细胞分离方法。方法:对用RosetteSep(法直接从全血分离NK细胞的方法进行改进,先从全血中获得单个核细胞(PBMC),与红细胞按比例混合后再进行分离,利用流式细胞仪检测纯度,并且用CCK-8法检测NK细胞对K562细胞的杀伤活性。结果:改进后分离每毫升血所需RosetteSep抗体复合物用量仅为直接分离法的1/50;NK细胞的纯度由纯化前的(11.02±3.15)%提高到(96.52±2.42)%,细胞回收率为(70.24±10.51)%;纯化的NK对靶细胞K562有较强的杀伤作用,与直接分离法比较杀伤活性差异无统计学意义(P>0.05)。结论:改进后的方法可以获得高纯度的NK细胞,该分离方法不影响NK的杀伤活性,同时极大的降低了分离成本。初步建立了一种经济高效,快速简便,并且纯化率高的人外周血NK细胞纯化方法。

Objective：To establish an effective method to obtain NK cells with high purity at low cost. Methods： We modified the method to obtain NK ceils directly from fresh whole blood by RosetteSep method, prior to addition of the bifunctional antibody cocktail for NK cell enrichment, PBMC were harvested from the Ficoll - Hypaque/plasma interface, and RBC was added to the PBMC suspension at a ratio of 100 ： 1. The proportion of NK cells was determined by flow cytometry and NK cells activity against the leukemic cell line K562 tested by CCK -8. Results ：The modified method reduced the cost of NK cells isolation by a factor of 50 compared with directly using whole blood. NK cells were purified from （ 11.02 ± 3.15 ） % of peripheral blood lymphocytes to a final of（ 96.52 ± 2.42 ） % , the recovery of NK cells was （70.24 ± 10.51 ）%, the purified NK cells demonstrated high cytotoxic activity against K562 target cell. Conclusion：The modified method is time -saving and effective to obtain NK cells with high purity at low cost.