[Comparative Study of the Two High-Efficient Strategies for in vitro Generation of Human Umbilical Cord Blood-derived Natural Killer Cells].

To explore the similarities and variations of biological phenotype and cytotoxicity of human umbilical cord blood natural killer cells (hUC- NK) after human umbilical cord blood-derived mononuclear cells (hUC-MNC) activated and expanded by two in vitro high-efficient strategies.Umbilical cord blood mononuclear cells (MNC) from healthy donor were enriched by Ficoll-based density gradient centrifugation. Then, the phenotype, subpopulations, cell viability and cytotoxicity of NK cells derived from Miltenyi medium (denoted as M-NK) and X-VIVO 15 (denoted as X-NK) were compared using a "3IL" strategy.After a 14-day's culture, the contents of CD3-CD56+ NK cells were elevated from 4.25%±0.04% (d 0) to 71%±0.18% (M-NK) and 75.2%±1.1% (X-NK) respectively. Compared with X-NK group, the proportion of CD3+CD4+ T cells and CD3+CD56+ NKT cells in M-NK group decreased significantly. The percentages of CD16+, NKG2D+, NKp44+, CD25+ NK cells in X-NK group was higher than those in the M-NK group, while the total number of expanded NK cells in X-NK group was half of that in M-NK group. There were no significant differences between X-NK and M-NK groups in cell proliferation and cell cycle, except for the lower percentage of Annexin V+ apoptotic cells in M-NK group. Compared with X-NK group, the proportion of CD107a+ NK cells in M-NK group were higher under the same effector-target ratio (E∶T) (P<0.05).The two strategies were adequate for high-efficient generation of NK cells with high level of activation in vitro, however, there are differences in biological phenotypes and tumor cytotoxicity.体外高效活化扩增人脐带血来源自然杀伤细胞的 两种体系的比较研究.探讨人脐带血来源的单个核细胞（hUC-MNC），经两种体外培养体系活化扩增后的人脐带血自然杀伤细胞（hUC-NK）在生物学表型和细胞毒性上的异同.收集健康供者捐赠的脐带血，经淋巴细胞分离液介导的密度梯度离心法富集单个核细胞。基于本课题组近期建立的3因子体系（定义为“3IL”），比较Miltenyi和X-VIVO 15两种培养基体外培养14 d后NK细胞（分别定义为M-NK和X-NK）的细胞表型、细胞亚群、细胞活力及细胞毒功能的相似性和差异性.经上述2种体系诱导14 d后，总CD3-CD56+ NK细胞比例由4.25%±0.04% (d 0)提高至71%±0.18%（M-NK）和75.2%±1.1%（X-NK）。与X-NK组相比，M-NK组CD3+CD4+ T和CD3+CD56+ NKT细胞比例显著降低。X-NK组中表达CD16、NKG2D、NKp44、CD25活化型标志物的细胞比例更高，但M-NK组总NK细胞扩增数量为X-NK组的2倍。两组在NK细胞增殖及细胞周期上无统计学差异，M-NK组的Annexin V+凋亡细胞比例低于X-NK组。NK细胞与K562细胞体外共培养杀伤实验显示，在相同效靶比（E∶T）下，M-NK组CD107a+ NK细胞的比例更高，杀伤效率亦略优于X-NK组（P<0.05）.两种体外扩增活化体系均可诱导产生高活化水平的hUC-NK，但二者在生物学表型和肿瘤杀伤毒性等多个方面存在差异.