不同细胞因子组合对人自然杀伤细胞体外高纯度扩增的实验研究

The experimental study on the expansion and high purity of human NK cells stimulated by different cy- tokines combination in vitro

目的探讨人自然杀伤细胞（NK）高效扩增培养技术，以获得一种优化的诱导扩增临床应用级别NK细胞的方法。方法提取健康人外周血单个核细胞（PBMCs），应用免疫磁珠分选法（MACS）阴性分离出高纯度的CD3-CD56＋CD16＋NK细胞，采用X-VIVO培养基，在均含有白细胞介素（IL）-2、CD3单抗的基础上用不同的细胞因子组合对其进行体外扩增和诱导。采用细胞计数法、流式细胞术免疫表型分析比较NK细胞增殖后的数量与NK细胞纯度。结果IL-7／IL-15／IL-21的优化组合刺激NK细胞增殖的效果为最佳，培养18d可使经免疫磁性珠阴性纯化的NK细胞扩增124倍，使未经纯化的PBMCs细胞增殖108倍。在NK细胞纯度方面，培养18d可使经免疫磁性珠阴性纯化的NK细胞扩增后的CD3-CD16＋CD56＋NK细胞纯度达81．5％，CD3-CD16＋CD56＋NK细胞阳性率为10．3％，NK细胞总纯度为91．8％；使未经纯化的PBMC细胞扩增后的CD3。CDl6＋CD56＋NK细胞纯度达22．1％，CD3＋CDl6＋CD56＋NK细胞阳性率为33．5％，NK细胞总纯度为55．6％。而单用不同细胞因子IL-7、IL-15、IL-21对NK细胞扩增数量和纯度均有所不同。结论采用X-VIVO培养基，在IL-2、CD3单抗的基础上加IL-7／IL-15／IL-21的优化组合刺激NK细胞增殖的数量和纯度达到最佳效果，所产生的NK细胞质量可满足临床细胞免疫治疗抗肿瘤应用的需要。

Objective This study explored the method of effective amplification of human natural killer （NK） cells in vitro, in order to obtain an optimized quality of NK cells for clinical applications. Methods CD3-CD16＋CD56＋NK cells were negatively selected using immunomagnetic bead sorting （MACS） from nor- mal peripheral blood mononuclear cells （PBMCs）. Both purified NK cells and PBMCs were cultured in X-VI- VO medium containing interleukin-2 （IL-2） and anti-CD3 monoclonal antibody . Various cytokines including IL- 7 ,IL-12 ,IL-15 and IL-21 were added to the culture in different combination. After 18 days of culture,the folds of NK cell expansion and the purity of NK cells were analyzed by cell counting and flow cytometry, respectively. Results After 18 days in culture and in the present of IL-2 and anti-CD3 MoAb,IL-7/IL-15/IL-21 in combi- nation was able to stimulate the purified NK cells to expand in 124 folds and PBMCs in 108 folds. The purity of CD3-CD16＋CD56＋NKand CD3-CD16＋CD56＋NK cells can reach to 81.5% and 10.3% ,respectively ,in the purified NK cells,while the purity of CD3-CD16＋CD56＋NKand CD3-CD16＋CD56＋NK cells can reach to 22.1% and 33.5% ,respectively,in PBMC cells. The number and purity of NK cells obtained by the addi- tion of cytokine IL-7,IL-15 or IL-21 individually were various. Conclusion Using X-VIVO medium containingIL-2,anti-CD3 MoAb and IL-7/IL-15/IL-21 in combination, we obtained the best effect of NK cell expansion and purity. The cell number,purity and quality of the NK cells produced by this method can meet the needs for cellular immunotherapy against tumors in clinical applications.