Li Xiao-Hong, Ma Jian, Wu Xiao-Xiong, Wang Fei-Fei, Li Meng, Da Wan-Ming, Yu Li, Gao Chun-Ji
Center of Hematology, PLA General Hospital, Beijing, China.
Zhonghua Xue Ye Xue Za Zhi. 2009 Jun;30(6):404-8.
To explore the expansion method of high purity NK cells from human peripheral blood and explore the changes in biological functions of NK cells after ex vivo expansion.
NK cells were isolated from peripheral blood mononuclear cells (PBMNCs) by using miniMACS (Magnetic cell-selection) and NK Cell Isolation Kit II, and cultured in SCEM (Stemline Hematopoietic Stem Cell Expansion Medium, Sigma) supplemented with 10% human AB serum and different combinations of interleukin (IL)-2 and/or IL-12, IL-15 for 15 days. Cultures were semi-exchanged with fresh media and cytokines every 3 days. Evaluation for cell expansion, phenotype, perforin and granzyme B mRNA expressions, and IFN-gamma secretion before and after the culture period.
CD3(-) CD56(+) cells concentration increased from (11.2 +/- 5.2)% to (94.2 +/- 3.5)%. In group IL-2 + IL-15 and IL-2 + IL-15 + IL-12 group, cells were expanded 50.5 +/- 4.3 and 52.3 +/- 6.7 - fold, respectively, being significantly higher than that in other three groups [(15.4 +/- 1.1 fold in IL-2 group, 19.9 +/- 3.9 fold in IL-2 + IL-12 group, 6.1 +/- 1.0 fold in control group)] (P<0.01), but no significant difference between each other (P>0.05). The purity of CD3(-) CD56(+) NK cells was over 94% in all groups except the control. The perforin and granzyme B mRNA expressions of expanded NK cells in four experimental groups were significantly higher than those of before expansion (P<0.01) and the expressions in IL-2 + IL-15 and in IL-2 + IL-12 + IL-15 group were significant higher than in other three groups (P<0.01) while no significant difference between each other (P>0.05). IFN-gamma levels in the supernatants of four experiment groups were significantly higher than that in control group (P<0.01) and its levels order was IL-2 + IL-15 + IL-12 group > IL-2 + IL-12 group > IL-2 + IL-15 group > IL-2 group (P<0.01).
High purity NK cells isolated by negative selection using miniMACS can be efficiently expanded with IL-2 + IL-15, and their biological functions were enhanced.
探索从人外周血中扩增高纯度自然杀伤（NK）细胞的方法，并探讨体外扩增后NK细胞生物学功能的变化。
采用miniMACS（磁性细胞分选）和NK细胞分离试剂盒II从外周血单个核细胞（PBMNCs）中分离NK细胞，在添加10%人AB血清以及白细胞介素（IL）-2和/或IL-12、IL-15不同组合的SCEM（Stemline造血干细胞扩增培养基，Sigma）中培养15天。每3天用新鲜培养基和细胞因子进行半量换液。评估培养前后细胞的扩增情况、表型、穿孔素和颗粒酶B mRNA表达以及干扰素-γ分泌情况。
CD3(-)CD56(+)细胞浓度从(11.2±5.2)%增至(94.2±3.5)%。在IL-2 + IL-15组和IL-2 + IL-15 + IL-12组中，细胞分别扩增了50.5±4.3倍和52.3±6.7倍，显著高于其他三组[IL-2组为15.4±1.1倍，IL-2 + IL-12组为19.9±3.9倍，对照组为6.1±1.0倍]（P<0.01），但两组之间无显著差异（P>0.05）。除对照组外，所有组中CD3(-)CD56(+)NK细胞的纯度均超过94%。四个实验组中扩增后的NK细胞穿孔素和颗粒酶B mRNA表达均显著高于扩增前（P<0.01），且IL-2 + IL-15组和IL-2 + IL-12 + IL-15组的表达显著高于其他三组（P<0.01），但两组之间无显著差异（P>0.05）。四个实验组上清液中的干扰素-γ水平均显著高于对照组（P<0.01），其水平顺序为IL-2 + IL-15 + IL-12组>IL-2 + IL-12组>IL-2 + IL-15组>IL-2组（P<0.01）。
采用miniMACS阴性分选法分离的高纯度NK细胞可通过IL-2 + IL-15高效扩增，且其生物学功能增强。
