细胞因子诱导的杀伤细胞体外扩增及其生物学特性的研究

The study of the expansion in vitro and its biological characteristics of cytokine-induced killer cells

目的:观察细胞因子诱导的杀伤细胞(CIK)的体外增殖能力及其生物学特性。方法:取正常成人新鲜血,经密度梯度离心法,利用连续非贴壁的方法获得单个核细胞(PBMC),加入不同的细胞因子(IFN-γ、IL-1β、IL-2、CD3mAb),诱导生成CIK细胞,以LAK细胞作为对照。观察CIK细胞的扩增情况,用流式细胞仪检测其表面标志,MTT法测定其杀瘤活性。结果:诱导16d后,CIK细胞的增殖率达到高峰,具有较强的扩增能力,经过22d培养,总的细胞数可扩增至100多倍;CIK细胞和LAK细胞相比,在前期无明显差异,至18、21d时扩增能力显著高于LAK细胞的扩增倍数(P<0.01)。CIK细胞是一群异质细胞群,CD3+CD56+细胞为其主要的效应细胞,经过22d培养显著增加,为(55.15±5.49)%,其绝对值可增加1500多倍。CIK对A-549细胞的杀伤活性均显著高于LAK细胞(P<0.05),具有较强的杀瘤能力。结论:CIK细胞具有较强的扩增和杀瘤能力,是肿瘤过继免疫治疗中更为有效的杀瘤效应细胞。

Objective： To observe the proliferation ability in vitro and its biological characteristics of cytokine-induced killer cells. Methods： PBMCs were isolated with successive non-adhere method from adult venous blood ,then were induced to be eytokine-indueed killer cells by fieoll density gradient centrifugation in the presence of IFN-γ、IL-1β、IL-2 and anti-CD3 antibody. LAK （lymphokine activated killer） cells were prepared as control. The proliferation ability of CIK cells was measured by calculation, the phenotype of CIK cells was analyzed by flow cytometry（FCM）and the killing tumor cell activity of CIK cells was tested with MTT method. Results： After 56 days of induction the proliferation rate of CIK cells reached a high peak, possessing strong proliferate ability with over 500 times expansion after 25 days culture; In comparison with LAK, CIK cells had no difference in early stage, but onday 58,21 the multiplication ability of CIK cells was higher than that of LAK cells markedly （P〈0.05）. CD3＋CD56＋ cells, the major effect cell in CIK cells group, had a significant increase in both absolute number and the percentage rate. The absolute unmber of CD3＋CD56＋ cells reached over 5 500 times and the percentage was 55.15% after being cultivated for 25 days culture. The killing activity of CIK cells were significantly stronger than that of LAK cells when A-549 cells was used as target cells （P〈0.05）. Conclusion： CIK cells have strong proliferation ability and killing activity,which were more efficient killer cell in the therapy of tumor adoptive immunity.