免疫杀手细胞的体外扩增方法及其对肝癌细胞BEL-7402的杀伤作用

Evaluation of a modified culture method for immune-killer cells and therapeutic efficiency on hepatocarcinoma cell line BEL-7402

目的:探讨免疫杀手细胞(immune-killer cells,IKCs)的体外扩增方法及其对人肝癌细胞BEL-7402的杀伤作用。方法:改良自然杀伤细胞培养扩增法扩增IKCs细胞,流式细胞仪分析IKCs表型,台盼蓝染色计数法观察细胞增殖状况,AlamarBlue法观察IKCs细胞对BEL-7402细胞的杀伤作用,用BALB/c裸鼠模型检测IKCs细胞对肝癌动物模型的治疗作用。结果:IKCs细胞培养14 d的细胞总数扩增300倍以上,主要由CD3^+CD56^+、CD3^-CD56^+和CD8^+细胞亚群组成,CD3^+CD56^+和CD3^-CD56^+比例达85%以上,CD8^+比例达到77%以上。IKCs细胞对肝癌BEL-7402细胞的杀伤实验显示,当效靶比为25:1时和100:1时,其杀伤活性分别为66.5%和97.8%(P<0.01)。裸鼠肝瘤模型治疗实验显示,IKCs细胞对早期和晚期人肝癌BEL-7402细胞裸鼠模型均有显著的治疗作用,与对照组比较,其抑瘤率达69%以上(P<0.01)。结论:本实验建立的肿瘤免疫杀手细胞IKCs的培养扩增方法是可行的、有效的,有望成为一种新的肿瘤免疫细胞治疗方法。

OBJECTIVE：To evaluate the modified culture method and the therapeutic efficiency of immune-killer cells（IKCs） on a hepatocarcinoma cell line BEL-7402.METHODS：IKCs were induced and amplified from PBMC cells in a modified NK culture method.The immunophenotype changes were analyzed by flow cytometry.The proliferation of the cells were evaluated by Trypan-blue staining.The killing activities on BEL-7402 cell line were detected by Alamar Blue staining and their effect on BEL-7402 tumor in the nude mice model were examined by monitoring tumor growth.RESULTS：The IKCs cultured for 14 days multiplied approximately 300 times.Cell subsets of CD3~＋CD56~＋,CD3^-CD56~＋ and CD8~＋ were the major components.Both ratios of CD3~＋CD56~＋ and CD3~CD56~＋ were higher than 85%,while the ratio of CD8~＋ was higher than 77%.At the effector-target ratio of 25：1 and 100：1,the killing activity against BEL-7402 cells of IKCs was 66.5%（P0.01） and 97.8%（P0.01）,respectively.Treatment with IKCs on BEL-7402 tumor in nude mice showed a noticeable higher therapeutic effect than that of control,with an inhibition ratio on tumor growth of 69%（P 0.01）.CONCLUSION：The modified culture method for IKCs was practicable and effective.The research might provide experimental basis for clinical immunotherapy of IKCs on hepatocarcinoma.