以肝素和抗CD16抗体为基础的扩增人脐血NK细胞无血清培养体系的建立

Establishment of A Serum-Free Culture System Based on Heparin and Anti-CD16 Antibody for Expansion of Human Cord Blood NK Cells

目的:建立一种新方法以用于体外培养扩增人脐血来源的NK细胞,从而为NK细胞的治疗奠定基础。方法:收集人脐血单个核细胞,将细胞按1.5×10~6/ml悬浮于含5%自体血浆、重组人IL-15(50 ng/ml)和氢化考的松琥珀酸钠(5×10^(-8)mol/L)组成的无血清培养液中,接种于含肝素钠或/和重组抗人CD16单克隆抗体铺板(预先铺板)的培养瓶中,其剂量分别为100 U/cm^2和1μg/cm^2底面积。培养2周后收集细胞,计细胞总数,应用流式细胞术检测细胞中CD3^-/CD56^+比例。以K562细胞为靶细胞,应用MTT实验观察不同效靶比条件下的NK的细胞毒作用。结果:与未铺板培养体系比较,抗CD16抗体及肝素钠铺板体系培养的细胞,在培养1周内易见贴壁的集落。培养2周后,预先铺板组细胞扩增倍数明显高于未铺板组,且CD3^-/CD56^+比例显著升高,尤以抗体和肝素混合铺板组为显著(P<0.01)。MTT实验显示,预先铺板培养组所获得细胞对K562细胞的杀伤活性更强。结论:利用肝素和抗CD16抗体铺板,在培养体系中加入IL-15和氢化考的松,可使NK细胞优势扩增,从而获得纯度较高、细胞毒活性较强的NK细胞。

Objective： To establish a novel method for ex vivo expansion of natural killer cells from human umbilical blood, so as to provide the basis for NK cell therapy. Methods： Mononucleated cells from human umbilical blood were harvested and suspended in a serum-free medium containing 5% autologous plasma, recombinant human IL-15 （50 ng/ ml） and hydrocortisone sodium succinate （5×10^-8 mol/L） at a concentration of 1.5×10^6/ml, then the cells were seeded into flasks pre-coated with heparin sodium （ 100 U/cm2 ） or/and anti-human CD16 antibody （ 1μg/cm2 ）. After culture for 2 weeks, the cells were harvested and counted. Ratios of CD3 -/CD56＋ of the cells were determined by flow cytometry. MTT test was performed to assess the cytotoxicity against K562 cells with graded ratios of effector/target cells. Results： In contrast to the cells in flasks without pre-coating, the attached colonies appeared predominantly within 1 week of culture from heparin - and antibody - coated groups. The cell numbers from the pre-coated groups were significantly higher than that of uncoated one after culture for 2 weeks. Furthermore, the ratios of CD3 -/CD56 ＋ cells were much higher in pre-coated groups, and that of the cells from flasks pre-coated with heparin and antibody were the highest （all the P values 〈0. 01 ）. MTT test showed that the cytotoxic activity of the cells stimulated by precoating were much more potent than that of the cells without the stimulation. Conclusion ： Advantageous expansion of NK cells can be achieved by precoating with heparin and anti-CD16 antibody, and also by supplement with IL-15 and hydrocortisone into the media, so the umbilical NK cells with high purity and potent cytotoxicity can be obtained.