IL-15与IL-21细胞因子组合优化临床级人NK细胞培养体系  

Optimization of clinical-grade human NK cell culture system with the combination of IL-15 and IL-21 cytokines

作　　者：陈伟[1] 杨冀[1] 蔺迪[1] 刘军德[1] 欧阳明玥 薛峰[1] 孔伟圣 于卉影[1] 

机构地区：[1]沈阳军区总医院全军肿瘤诊治中心医学实验科,辽宁沈阳110016 [2]珠海贝索细胞科学技术有限公司,广东珠海519015

出　　处：《临床军医杂志》2017年第1期64-68,72,共6页Clinical Journal of Medical Officers

摘　　要：目的探讨IL-15、IL-21细胞因子组合建立的临床级人NK细胞培养体系对NK细胞增殖、免疫细胞表型和细胞毒作用的影响。方法分离9例健康志愿者外周血单个核细胞(PBMCs),根据以下3组进行扩增培养:(1)对照组(我科临床级人NK细胞培养体系),(2)IL-15组(对照组+IL-15),(3)IL-15/IL-21组(对照组+IL-15+IL-21)。台盼蓝拒染法计数细胞总数和活力;流式细胞术分析免疫细胞表型和NK细胞活化受体表达;乳酸脱氢酶释放法检测细胞毒活性。结果培养至14 d,IL-15/L-21组细胞总数明显升高,NK细胞增殖倍数达到(761.68±449.30)倍,明显高于IL-15组,差异有统计学意义(P<0.05);IL-15/IL-21组明显高于对照组,差异有统计学意义(P<0.01)。3组培养物中CD3^-/CD56^+细胞百分率比较,IL-15/IL-21组和IL-15组明显高于对照组,差异有统计学意义(P<0.05),但IL-15/IL-21组和IL-15组比较,差异无统计学意义(P>0.05);3组中CD3^+/CD4^+细胞百分率均呈下降趋势,且IL-15/IL-21组和IL-15组明显低于对照组,差异有统计学意义(P<0.05),但IL-15/IL-21组和IL-15组比较,差异无统计学意义(P>0.05);3组中总CD3^+细胞百分率比较,IL-15组明显低于对照组(P<0.05),但IL-15/IL-21组和IL-15组比较,差异无统计学意义(P>0.05)。IL-15/IL-21组NKG2D活化受体表达水平明显高于对照组,差异有统计学意义(P<0.05),但与IL-15组比较,差异无统计学意义(P>0.05)。3组中CD3^+/CD56^+细胞百分率、CD3^+/CD8^+细胞百分率以及NKp46活化受体的表达水平比较,差异均无统计学意义(P>0.05)。相同的效靶比,3组细胞的细胞毒作用比较,差异均无统计学意义(P>0.05)。结论采用IL-15/IL-21组合优化人NK细胞培养体系,可以选择性地扩增NK细胞,提高培养物中NK细胞数量和细胞总数,以及活化受体NKG2D的表达水平,这将为优化临床级人NK细胞制备体系提供科学依据。Objective To investigate the effect of the combination of IL-15 and IL-21 cytokines on proliferation,immune cell phenotype and cytotoxicity of the cells in a clinical-grade culture system of human NK cell. Methods Peripheral blood mononuclear cells（ PBMCs） isolated from 9 healthy volunteers were induced and amplified in the following 3 groups in vitro：（ 1） control group（ the clinical grade human NK cell culture system of our lab）,（ 2） IL-15 group（ control group ＋ IL-15）,（ 3） IL-15 / IL-21 group（ control group ＋IL-15 ＋ IL-21）. The cell number and viability were performed by trypan blue exclusion; cell phenotypes and NK activating receptors were analyzed with flow cytometry with the samples collected on Day 0 and 14. The cytotoxicity of cultured cells were determined by lactate dehydrogenase（ LDH） release assay on day 14. Results At the end of culture（ Day 14）,the total cell number of IL-15 / IL-21 group was significantly increased（ P〈0. 05）,in particular the NK cell proliferation rate reached a fold expansion of（ 761. 68 ±449. 30） times,significantly higher than those of IL-15 group（ P〈0. 05） and control group（ P〉0. 01）. IL-15 / IL-21 group and IL-15 group can significantly increase the CD3^-/ CD56^＋ cell percentage of NK cells in culture than that of control group（ P〈0. 05）,while there were no significant differences between IL-15 / IL-21 group and IL-15 group（ P〈0. 05）; IL-15 / IL-21 group and IL-15 group can significantly decrease the percentage of CD3^＋/ CD4^＋ cells in culture than that of control group（ P〈0. 05）,there were no significant differences between IL-15 / IL-21 group and IL-15 group（ P〈0. 05）; IL-15 group can significantly decrease the percentage of all CD3^＋ cells in culture than that of control group（ P〈0. 05）,while there were no significant differences between IL-15 / IL-21 group and IL-15 group（ P〈0. 05）. The expression level of NKG2 D activating receptor was higher in IL-15 / IL-21 gro

关 键 词：自然杀伤细胞 免疫治疗 白细胞介素-15 白细胞介素-21 抗人表皮生长因子受体2单克隆抗体 细胞培养 

分 类 号：R373.2[医药卫生—病原生物学]