细胞因子组合体外扩增人NK细胞的研究

Amplification of human NK cells in vitro with combinations of interleukin

目的:探讨细胞因子IL-2、IL-12、IL-15和IL-18组合对体外扩增人外周血来源的NK细胞受体表达及杀伤肿瘤细胞能力。方法:NK细胞的培养分为CIK组、IL-2+IL-15组和IL-2+IL-12+IL-15+IL-18组;流式细胞术检测培养的细胞表型及NK细胞受体表达;LDH法检测不同效靶比NK细胞对不同肿瘤细胞株的杀伤作用。结果:外周血淋巴细胞经细胞因子IL-2+IL-12+IL-15+IL-18组合作用下,17d后NK细胞(CD3-CD56+)比例上升至80%以上,NK细胞数扩增>1 000倍,明显高于其他两组,F=37.154,P<0.001。NK细胞活化标志CD69+分子增加(t=21.271,P<0.001),NK细胞活化性受体(NKG2D、NKp30、NKp44和NKp46)均有不同程度上调(P值均<0.05),而NK细胞抑制性受体CD158b(t=3.416,P=0.021)和CD159a(t=4.209,P=0.018)不同程度下调,T淋巴细胞、B淋巴细胞(CD19+)、单核巨噬细胞(CD14+)、调节性T细胞(T-reg)等均显著减少,P<0.001。对扩增的NK细胞杀伤敏感的肿瘤细胞株均高表达MHCⅠ类相关蛋白A(major histocompatibility complex classⅠchain related molecules A,MICA),MICA表达分别为K562(46.2±3.2)%、MCF-7(56.5±4.7)%、HTC-8(52.5±4.1)%和Eca-109(36.5±2.5)%,而对NK细胞抵抗的细胞株均低表达或不表达MICA分子,分别为Raji 0、MDA-MB-435s0和HT-29(1.2±0.8)%。结论:细胞因子IL-2+IL-12+IL-15+IL-18组合能有效的扩增外周血来源NK细胞,上调其活化性受体,下调抑制性受体,其数量及功能均满足临床治疗需要。

OBJECTIVE： To explore the changes in biological functions of human peripheral blood derived NK cells after ex- ternal expansion with combinations of i nterleukin IL- 2, IL- 12, IL- 15 and IL- 18. METHODS .. Cultured NK cells were divided into 3 groups： group CIK, group IL-2 ＋ ILl 5 and group IL-2 ＋ IL- 12 ＋ IL- 15 ＋ IL- 18. μK cell recepto expressions were determined by flow cytometry. The cytotoxicity of cultured NK cells to kinds of tumor lines were tested by LDH. RESULTS.. The proportion of NK cell markers CD3-CD56＋ increased to more than 80% after 17 days cultured in group IL-2-bIL-12＋IL-15＋IL-18,meanwhile,NK cells was amplifiedl 000-fold, significantly higher than that in the other two groups （F： 37. 154, P〈0. 001）. After cultured, NK cells activation marker CD69＋ molecule increasd （t=21. 271,P〈0. 001） and NK cells activating receptor （NKG2D, NKp30, NKlyI4, NKp46） was showed in different levels of increase（all P〈0. 05） and inhibitory receptor （CD158b,CD159a） was decreased （t=3. 416, P=0. 021;t=4. 209,P=0. 018）. T cells,B cells （CD19） ,monocyte-macrophage cells （CD14） ,regulatory T cells （T-reg） were in a significant reduction （P〈0. 001）. High expression of major histocompatibility complex class I of chain related molecules A molecules （MICA） were sensitive to NK cells cytotoxicity, respectively for K562 （46.2±3. 2）% ,MCF-7 （56.5±4. 7）%, HTC-8 （52. 5±4. 1） % ,Eca-109 （36.5±2.5）% ,and 10w or no expression of MICA molecules were resistance to NK cells cytotoxicity, respectively for Raji 0,MDA-MB-435s 0,HT-29 （1.2±0. 8） %. CONCLUSIONS： The combination of IL-2＋IL-12＋IL-15＋IL-18 has synergistic effect on strengthening cytotoxicity of NK cells and promoting cell expansion, up-regulates its activated receptor. The number and function of the cultured NK cells can meet the clinical treatment needs.