利用滤除白细胞分离培养自然杀伤细胞方法的优化

Optimization of the method to cultivate NK cells from abandoned white cells

目的利用制备血液制品中滤除的白细胞，体外诱导扩增自然杀伤细胞（NK），并对NK细胞的分离培养方法进行优化。方法收集15例健康献血者的新鲜外周血，将制备血液制品中滤除的白细胞回收，经密度梯度离心法获得外周血单个核细胞（PBMCs），每份样本分为CD3mAb组（包被CD3单克隆抗体）、CD16mAb组（包被CD16单克隆抗体）和CD16mAb＋CD3mAb组（包被CD16单克隆抗体和CD3单克隆抗体）。相同培养条件下，加入相同无血清细胞培养液和细胞因子培养、诱导扩增。在显微镜下观察细胞扩增倍数，采用流式细胞术检测细胞表型CD3-CD56＋细胞比例、NK细胞表面活化性受体的表达及细胞杀伤因子干扰素γ（IFN-γ）的分泌，采用乳酸脱氢酶（LDH）释放法及流式细胞术检测NK细胞对肿瘤细胞的细胞毒作用。结果培养第17天，CD3mAb组、CD16mAb组和CD16mAb＋CD3mAb组的CD3-CD56＋细胞比例分别为（15．19±12．22）％、（83．63±10．63）％和（49．40±12．64）％，而培养前CD3-CD56＋细胞比例为（16．34±10．51）％。CD16mAb组和CD16mAb＋CD3mAb组CD3-CD56＋细胞比例与CD3mAb组CD3-CD56＋细胞比例比较，差异有统计学意义（P〈0．05）。CD3mAb组、CD16mAb组和CD16mAb＋CD3mAb组的NK细胞总数均〉10^9个。流式细胞术检测结果显示，CD16mAb组培养前CD3-CD56＋细胞表面活化性受体CD314、CD335、CD337的表达率分别为（68．59±5．01）％、（20．94±3．48）％和（33．27±6．27）％，培养第17天分别为（73．63±4．50）％、（57．53±4．77）％和（64．65±4．18）％，培养前后CD314表达率的差异无统计学意义（P〉0．05），培养前后CD335、CD337表达率的差异有统计学意义（P〈0．05）。扩增后的NK细胞对肿瘤细胞K562的细胞毒作用在效靶比为40：1时可达到（76．97±3．16）％。结论滤除的白细胞经体外分离培养后可得到比例高达90％的NK细胞，其细胞总数〉10^9个，细胞活性增强，对肿瘤细胞具有较高的杀伤活性，可达到临床治疗肿瘤的应用标准。

Objective Using abandoned white cells separated from preparation of blood products to cultivate NK cells in vitro, and to optimize the method of cultivation of allogeneic NK cells for clinical application. Methods Abandoned white cells separated from blood production were collected from 15 healthy donors. PBMCs were isolated from the abandoned white cells and cultured for 17 days using culture bottles as previously coated antibodies （group CD3 mAb was coated with CD3 mAb, group CD 16mAb was coated with CD16mAb, and group CD3 mAb＋CD16 mAb was coated with CD3 mAb and CD16 mAb）. Flow cytometry was used to determine the ratio of CD3- CD56＋ cells, expression of activated cell surface receptors, and secretion of IFN-γ. The anti-tumor cytotoxicity against K562 and Raji cells was determined using LDH cytotoxicity assay and flow cytometry. Results After expansion for 17 days, the proportions of CD3-CD56＋ cells was （15.19±12.22）% in the group CD3 mAb, （83.63±10.63）% in the group CD16 mAb, （49.40± 12.64）% in the group CD3 mAb＋CD16 mAb, and it was （16.34± 10.51）% before expansion. The total number of NK cells was more than 109. The expression ratios of NK cell surface activated receptors NKp30 and NKp46 were significantly increased, while that of the NKG2D was not significantly changed. The NK cells after expansion showed high cytotoxicity activity against K562 cells, reaching up to（76.97±3.16） % when effector-cell-to-target-cell ratio （E ： T ratio） was 40 ： 1. Conclusions NK cells can be obtained from abandoned white cells after cultivation for 17 days, with a purity up to 90% and total cell number of more than 109. Their activity was reinforced, the anti-tumor cytotoxicity activity was increased, and may meet the standard of clinical therapeutic application.