新疆地区健康成人外周血NK细胞体外扩增技术的研究

The study of NK cells’vitro amplification techniques in Xinjiang health adult blood

目的探索用免疫磁珠分选高纯度CD3-CD56+CD16+CD19-CD45+NK细胞并研究体外保持NK细胞活性及体外扩增技术。方法应用免疫磁珠阴性分选法(MACS)分离出高纯度的CD3-CD56+CD16+CD19-CD45+NK细胞,置于含10%胎牛血清的RPMI1640培养基中,分别加入500、1 000、6 000U/mL 3种不同浓度的细胞因子IL-2,组成不同培养体系进行体外扩增。采用细胞计数法、流式细胞术免疫表型分析方法,比较3种细胞因子刺激组及对照组培养的NK细胞数量及纯度的变化。结果免疫磁珠阴性分选后所得的高纯度CD3-CD56+CD16+CD19-CD45+NK细胞纯度由分选前的(29.66±2.31)%提高到(95.37±1.93)%,培养7d后,空白对照组NK细胞纯度降低为(61.82±2.58)%,而加入细胞因子IL-2的3组NK细胞纯度均无明显差异(P>0.05)。18d后,空白对照组的NK细胞纯度降低至(4.28±1.56)%,而浓度为6 000U/mL的实验组纯度为(93.72±2.29)%,与加入细胞因子IL-2前比较,差异无统计学意义(P>0.05)。NK细胞中加入3种不同浓度细胞因子IL-2组500、1 000、6 000U/mL未见明显扩增,差异无统计学意义(P>0.05)。结论经免疫磁珠分选法分选获得的NK细胞,在体外使用10%胎牛血清的RPMI1640培养基培养,添加刺激因子IL-2后,可有效提高NK细胞的存活率,为今后NK细胞的研究与免疫治疗提供了一种简单、有效保持NK细胞纯度的方法。

Objective The study aims to sort high purity CD3-CD56＋CD19＋CD16 CD45-NK cells in vitro, and to keep the activity of NK cells and in vitro amplification technique by means of immune magnetic bead. Methods By using immune magnetic bead negative sorting method （MACS）, separate CD3 ＋ CD16q-CD56-CD19＋CD45 NK cells with high purity, put them in bovine serum of RPMI1640 medium with 10% fetal, add three different concentration of cell factor IL-2 different training system of in vitro amplification. The cell count method, flow cytometry immune phenotypic analysis method are applied to compare the number of NK cells and the change of purity of three kinds of different concentration cell factors. Results CD3-CD56＋CD16＋CD19 CD45＋NK cells with high purity by Negative immune magnetic beads increase the purity from （29.66±2.31） % to （95.37±1.93） , after 7 dtraining, the purity in blank control group reduces to （61.82±2.58） %, NK cells with three groups of cytokine IL-2 NK have no significant differences （P〉0. 05）. After 18 d, the purity in blank control group of NK cells decreases to （4.28±1.56） 0/40, the purity of the group with concentration of 6 000 U/mL is （93.72±2.29） %, and there was no statistically significant difference （P 〉0.05）, compared with the cells before adding cytokines IL-2. NK cells with lhree different concentrations of cytokines IL-2 group of 500, 1 000, 6 000 U/mL did not show obvious amplification, so there was no statistically significant difference （P 〉0.05）. Conclusion The NK cells with immune magnetic bead sorting method, cultivated in vitro using 10% fetal calf serum RPMI1640 media by adding stimulating factor IL- 2 can effectively improve the survival rate of NK cells. A simple and effective method to keep the purity of NK cells is provided in future for the NK cells and immune therapy.