选择性扩增人自然杀伤细胞的实验研究

背景与目的:自然杀伤(naturalkiller,NK)细胞具有很强的杀瘤活性,但体外扩增NK细胞的数量难以达到治疗肿瘤的目的。本实验探讨由人外周血单个核细胞(peripheralbloodmononuclearcells,PBMCs)和附壁Wilms肿瘤细胞株HFWT混合培养能否有效地诱导NK细胞。方法:采用PBMCs和HFWT细胞混合培养诱导扩增NK细胞,51Cr释放法和结晶紫染色法测定NK细胞的杀瘤活性;流式细胞计数仪检测培养细胞中CD3 、CD4 、CD8 、CD16 和CD56 细胞的比例。结果:HFWT对人NK细胞特别敏感,它比不表达MHC-Ⅰ类分子的K562细胞和其他附壁生长的肿瘤细胞株更有效地刺激PBMCs,从而选择性扩增NK细胞。健康人PBMCs在HFWT细胞中培养10～21天后,CD56 CD16 细胞占细胞总数50%以上。当效靶比为2∶1时,NK细胞杀伤80%的HFWT细胞,NK细胞的扩增需要HFWT细胞反复刺激PBMC。结论:从HFWT细胞株中可选择性扩增人NK细胞,将有助于开展临床肿瘤过继免疫疗法。

Selective expansion of human natural killer cells

BACKGROUND & OBJECTIVE: Natural killer (NK) cells have strong potential of killing tumor cells, but it is difficult to expand sufficient NK cells to satisfy the treatment of tumor. This study was to explore the feasibility of efficiently expand NK cells through co-culturing peripheral blood mononuclear cells (PBMCs) and Wilms tumor cell line HFWT. METHODS: PBMCs and HFWT cells were co-cultured to expand NK cells. The cytotoxicity of NK cells was measured using 51Cr release assay and crystal violet (CV) staining. The proportions of CD3+, CD4+, CD8+, CD16+, and CD56+ cells were detected using flow cytometry. RESULTS:When PBMCs were co-cultured with irradiated HFWT cells for 10-21 days, NK cells were selectively expanded; the proportion of CD56+/CD16+ cells was over 50%. The NK cells killed more than 80% of fresh HFWT cells at an effector/target ratio of 2. The expansion of NK cells required continuous stimulation of PBMCs by HFWT cells. CONCLUSION: HFWT cells can effectively stimulating PBMCs to sensitively expand human NK cells for adoptive immunotherapy of human tumors.