利用单个核细胞培养扩增NK细胞体系探索

System Amplifying NK Cells Using Mononuclear Cell Culture

目的:探寻一种通过单个核细胞使自然杀伤(NK)细胞在体外培养扩增的新方法,为NK细胞免疫治疗奠定实验基础。方法:收集3份健康足月孕妇的脐带血,应用密度梯度法分离单个核细胞(PBMNC),每份单个核细胞样本均分为CD16 m Ab、CD3 m Ab和CD16 m Ab+CD3 m Ab处理3组。使用含自体血浆、重组人IL-2、IL-15和IL-21的无血清培养液,在相同条件下培养扩增14 d,计算其扩增倍数。应用流式细胞术测定CD56^+CD3^-比例;以K562细胞为靶细胞,应用MTT法测定所培养的NK细胞在不同效靶比条件下的杀伤率。结果:培养14 d后,CD16m Ab、CD3 m Ab和CD16 m Ab+CD3 m Ab组细胞总数分别扩增45.71±5.54、87.41±19.77和51.84±4.88倍,在CD3 m Ab组显著高于其它2组(P<0.05)。培养前CD56^+CD3^-细胞比例为0.1663±0.0201,经培养扩增14 d后CD16 m Ab、CD3 m Ab和CD16 m Ab+CD3 m Ab组CD56^+CD3^-细胞比例分别为0.8167±0.0500、0.8077±0.0589和0.8077±0.0273,3组之间无显著性差别。培养14 d后,MTT法检测显示,在效靶比为1∶1、5∶1和10∶1时3组杀伤效率均无显著差异(P>0.05)。结论:使用抗CD3 m Ab及IL-2,IL-15和IL-21培养单个核细胞,可获得高纯度和高杀伤活性的NK细胞。本研究为NK细胞的治疗提供了一个简单有效的方法。

Objective： To develop an easy method to amplify natural killer （NK） cells by using mononuclear cells in vitro, so as to lay the basis for NK cell therapy. Methods： Umbilical cord blood from 3 healthy full-term pregnant women was collected, and the peripheral blood mononuclear cells （PBMNC） were harvested by density gradient centrifugation. Each sample of PBMNC was divided into 3 groups： CD16mAb, CD3 mAb and CD16mAb ＋ CD3mAb- groups. The culture flasks were pre-coated with CD16, CD3 or CD3 plus CD16 mAb. The PBMNCs were cultured in serum-free media containing autologous plasma, recombinant human IL-2, IL-15 and IL-21 for 14 days under the same conditions. The total viable cell count was calculated. Flow cytometry was used to determine the ratio of CD56 ＊ CD3 cells, MTr assay was used to measure the killing rate of NK cells under different effector/target ratio, by using the K562 cells as the target cells. Results： After 14 days of culture, the total cell numbers of CD16mAb, CD3mAb and CD16mAb ＋ CD3mAb groups increased by 45.71±5.54, 87.41±19.77 and 4. 88±51.84 times, respectively, and those of CD3mAb group were significantly higher than the other 2 groups （ P 〈 0.05 ）. The ratio of CD56 ＋CD3 - cells before culture was 0. 1663 ± 0. 0201, which was 0. 8167±0. 0500, 0. 8077± 0. 0589 and 0. 8077±0. 0273 after incubation with CDI6mAb, CD3mAb and CD16mAb ＋ CD3mAb for 14 days, respectively （P 〉 0.05）. MTT test showed that the killing efficiencies were not significantly different among the 3 groups when the effector/target ratios were 1： 1, 5：1 and 10：1 （P 〉 0.05）. Conclusion： By incubation with anti-CD3 monoclonal antibody, IL-2, IL-15 and IL-21, the highly purified NK cells can be obtained from mononucleated cells, thus providing a simple method for NK cell therapy.