体外扩增高纯度的人外周血来源的NK细胞的研究

本研究探讨从人外周血中分选及扩增高纯度NK细胞的优化技术。先用miniMACS及阴性免疫磁珠分选方法,从外周血单个核细胞(PBMNC)得到纯化的NK细胞,随后在干细胞培液基条件下,通过IL-2、IL-12和IL-15等细胞因子的不同组合将培养体系分为IL-2组、IL2+IL12组、IL2+IL15组、IL2+IL15+IL12组和对照组(不加任何细胞因子),分别培养15天,每3天半量换液并补充细胞因子;检测分选及扩增前后(CD3-CD56+)NK细胞含量、扩增倍数及扩增前后各组在不同效靶比下的杀伤率。结果显示经:miniMACS阴性免疫磁珠分选后(CD3-CD56+)NK细胞含量由分选前的11.19±5.25%提高到94.23±3.50%。培养15天后除对照组(CD3-CD56+)NK细胞纯度略下降外,其余4组与扩增前无显著性差异(P>0.05)。在IL-2、IL2+IL12、IL2+IL15、IL2+IL15+IL12培养体系中,NK细胞扩增倍数分别为15.43±1.08,19.87±3.87,50.46±4.31和52.35±6.72,均显著高于对照组6.14±1.0(P<0.01),但IL2+IL15与IL2+IL15+IL12组间未见显著差异(P>0.05)。各组扩增的NK细胞对K562细胞的杀伤率均较扩增前增强,在不同效靶比下IL2+IL15、IL2+IL15+IL12组对K562细胞的杀伤率显著高于其他组,但两组间无显著性差异(P>0.05)。结论:经miniMACS免疫磁珠阴性分选得少量NK细胞后,应用IL2+IL15培养条件是获得高纯度NK细胞的简单有效方法。

Ex Vivo Expansion of Highly Purified NK Cells from Human Peripheral Blood

Adoptive immunotherapy using allogeneic natural killer (NK) cells provides to be useful in recipients after allogeneic hematopoietic stem cell transplantation (Allo-HSCT), but its application has been limited by the inability to obtain sufficient numbers of pure NK cells. This study was aimed to optimize the expansion of high purity NK cells from human peripheral blood. First, the NK cells were isolated from PBMNC by using miniMACS (magnetic cell-selection) and NK Cell Isolation Kit II. Then the isolated cells were cultured in SCEM (Stemline Hematopoietic Stem Cell Expansion Medium, Sigma) supplemented with 10% human AB serum and different combinations of IL-2 and/or IL-12, IL-15 for 15 days. Cultures were fed with fresh media and cytokines every 3 days, and were evaluated for cell expansion, phenotype, and cytotoxicity at the end of the culture period. The results showed that in group IL2 + IL15 and IL2 + IL15 + IL12, cells were expanded 50.46 +/- 4.31 and 52.35 +/- 6.72-fold respectively, much higher than others (P<0.01), but no significant difference between themselves (P>0.05). And the purity of CD3(-)CD56(+) NK cells was over 94% in all groups except the control. The cytotoxicity of expanded NK cells cultured with cytokines was significantly higher than the starting population at different E:T ratio (P<0.01), although the cytotoxicity of IL2 + IL15 + IL12 group was slightly higher than that of IL2 + IL15 group, but no significant difference between themselves (P>0.05). It is concluded that high purity of NK cells can be efficiently expanded in culture with IL2 + IL15.