NK细胞的高效扩增及其对体外肿瘤模型的杀伤效果研究

Efficient amplification of NK cells and their anti-tumor cytotoxic activity in vitro

目的:高效扩增NK细胞,并确定其对不同肿瘤的杀伤作用,为临床应用提供参考。方法:从成人外周血中分离PBMCs并利用表面表达4-1BBL、IL-15和IL-21的K562滋养细胞对其进行共刺激培养,15 d后计数并检测CD3^-CD56^+细胞纯度;利用间接免疫荧光及Real-time PCR方法检测NK细胞Granzyme B及Perforin表达变化的同时检测其对肺癌、胃癌、肝癌、卵巢癌、胰腺癌、宫颈癌的杀伤作用,确定其对不同肿瘤的杀伤效果。结果:扩增15 d后,细胞数量高于110亿,CD3^-CD56^+比例达95%以上;培养后的NK细胞高表达Granzyme B、Perforin,且对A549的杀伤效果(E∶T=10∶1)约为90%,同时对其他肿瘤细胞(E∶T=10∶1)的杀伤效果由强到弱的顺序为:胃癌、胰腺癌、宫颈癌、卵巢癌、肾癌、肝癌(P<0.05);NK细胞对宫颈癌、肝癌、胰腺癌的杀伤效果呈现一定的时间依赖性。结论:此方法可以扩增出高数量、高纯度的NK细胞,同时该NK细胞具有高效杀伤多种肿瘤细胞的能力。

Objective ： To efficiently amplify NK cells and deter^nine their cytotoxic activity against a variety of tumor cell lines in vitro,thereby providing evidence for potential clinical application. Methods ： PBMCs were isolated from adult peripheral blood and co-cultured with K562 cells that were genetically modified to express 4-1BBL, IL-15 and IL-21 on the surface for 15 days to effectively amplify NK cells. The total cell number and Purity of CD3-CD56＋ cells were measured. Granzyme B and perforin expression of the amplified NK cells were detected by flow cytometry and real-time PCR. The anti-tumor effect on different cancer cells was evaluated. Results： This method obtained a more than 1. 1x1010 CD3- CD56＋ NK cells with 95% purity over a 15 day amplification pro-cedure. The expanded NK cells could efficiently release granzyme B and Perforin. The cytotoxicity against different tumor cells was followed the order from strong to weak ： gastric, pancreatic, cer^^ical, ovarian and renal cancer cells, with the highest activity against gastric cancer cell line A549 （90% at E：T = 10 ：1） （P〈0. 05）. A time-dependent killing effect of activated NK cells on cervical,liver and pancreatic cancer cells was observed. Conclusion ： This amplification procedure can consistently generate large amounts of pure NK cells with effective cytotoxic function against a variety of tumor cells.