摘要
目的:探讨人外周血来源的单个核细胞经三种体外培养体系活化扩增后的自然杀伤细胞(NK细胞)在扩增倍数、生物学表型和细胞毒性上的异同.方法:收集健康供者捐赠的外周血,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.使用含自体血浆、重组人细胞因子的无血清培养液,各体系均添加IL-2和IL-15,体系B额外添加抗人CD3单抗,体系C额外添加IL-18,在相同条件下培养扩增14 d,比较不同培养体系的NK细胞在细胞表型、细胞活力及细胞毒性的异同.结果:体系A、B、C中NK细胞扩增倍数分别为811.43±30.07、2800.79±36.42和1047.97±85.21,表明抗人CD3单抗可以增强NK细胞的扩增.三种体系培养NK细胞在细胞表型、细胞周期、细胞活力和细胞毒性方面无统计学差异.结论:使用含自体血浆、重组人IL-2、IL-15细胞因子和抗人CD3单抗的无血清培养液,在 37℃、5%CO2 条件下可以实现NK细胞大规模培养,获取高纯度的NK细胞.
Abstract
Objective:To investigate the similarities and differences in amplification multiples,biological phenotypes and cytotoxicity of human natural killer cell(NK cell)derived from human peripheral blood mononuclear cells activated and expanded by three in vitro culture systems.Methods:Collect peripheral blood donated by healthy donors and enrich mononuclear cells using density gradient centrifugation mediated by lymphocyte separation solution.Using serum free culture medium containing autologous plasma and recombinant human cytokines,IL-2 and IL-15 are added to each system,anti-human CD3 monoclonal antibody is added to system B,and IL-18 is added to system C.Culture and amplify NK cells under the same conditions for 14 days,and compare the differences in cell phenotype,cell viability,and cytotoxicity of NK cells from different culture systems.Results:In three in vitro culture systems,the amplification ratio of NK cells in the system A,system B and system C are 811.43±30.07,2800.79±36.42,and 1047.97±85.21,respectively,which means anti-human CD3 monoclonal antibodies can enhance the amplification of NK cells.There are no statistically significant differences in cell phenotypes,cell cycle,cell viability,and cytotoxicity among the three systems for cultivating NK cells.Conclusion:Using a serum-free culture medium containing autologous plasma,recombinant human IL-2,IL-15 cytokines,and anti-human CD3 monoclonal antibodies,large-scale NK cell culture can be achieved at 37℃and 5%CO2 to obtain high-purity NK cells.
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