IL -15、IL-21和4-1BBL 增强NK-92细胞增殖和细胞毒性的实验研究  

IL-15,IL-21 and 4-1BBL enhance the proliferation and cytotoxicity of NK-92 cells

作　　者：闵静婷 黄艳平 樊红莲 赵报 汪洪涛[3] 李正红[1] Min Jingting;Huang Yanping;Fan Honglian;Zhao Bao;Wang Hongtao;Li Zhenghong(Department of Pathophysiology,Bengbu Medical College,Anhui Bengbu 233030,China;Department of Experiment Center,the First Affiliated Hospital,Bengbu Medical College,Anhui Bengbu 233004,China;Department of Infection and Immunity,Bengbu Medical College,Anhui Bengbu 233030,China)

机构地区：[1]蚌埠医学院病理生理实验室,安徽蚌埠233030 [2]蚌埠医学院第一附属医院实验中心,安徽蚌埠233004 [3]蚌埠医学院感染与免疫实验室,安徽蚌埠233030

出　　处：《现代肿瘤医学》2020年第13期2189-2193,共5页Journal of Modern Oncology

基　　金：国家自然科学基金(编号:81472656);安徽省重点研究和开发计划项目(编号:201904a07020019);蚌埠医学院2019年度研究生科研创新计划(编号:Byycx1912)。

摘　　要：目的:探讨IL-15、IL-21和4-1BBL组成的不同培养体系,对体外扩增的NK-92细胞毒活性的影响。方法:以三质粒系统包被携带IL-15、IL-21和4-1BBL基因的慢病毒,用慢病毒感染的方式制备2组饲养层细胞;筛选后经MMC处理分5组体外扩增NK-92细胞(NK-92组:NK-92细胞;K562组:K562细胞+NK-92细胞;IL-15组:表达IL-15和4-1BBL的K562细胞+NK-92细胞;IL-21组:表达IL-21和4-1BBL的K562细胞+NK-92细胞;IL-15+IL-21组:表达IL-15和4-1BBL的K562细胞、表达IL-21和4-1BBL的K562细胞+NK-92细胞);通过CCK-8法,乳酸脱氢酶释放法检测体外扩增后NK-92细胞的细胞毒性;ELISA检测NK-92细胞上清中IFN-γ的水平,以评估NK-92细胞的抗肿瘤效应。结果:两组转染后的HEK293FT细胞以及慢病毒感染的K562细胞携带绿色荧光,两组饲养层细胞成功表达目的基因;各组饲养层细胞都能刺激NK-92细胞大量增殖;随着效靶比的逐渐增加,IL-15组扩增后的NK-92细胞毒性显著高于其余各组(P<0.05);效靶比为5∶1和10∶1时,IL-15组NK细胞IFN-γ的释放量显著高于其余各组(P<0.05)。结论:膜结合型IL-15、IL-21与4-1BBL联合都能引起NK-92细胞的大量增殖,且IL-15与4-1BBL联合运用扩增的NK-92细胞有更强的杀伤肿瘤细胞的能力。Objective:To investigate whether the proliferation and cytotoxicity of NK-92 cells can be improved by the various culture system was composed of IL-15,IL-21 and 4-1BBL in vitro.Methods:Lentivirus carrying IL-15,IL-21 and 4-1BBL genes was coated with a 3-plasmid system,and 2 groups of feeder cells were prepared by lentivirus infection.After screening,NK-92 cells were amplified in vitro by MMC treatment into 5 groups(NK-92 group:NK-92 cells.K562 group:K562 cells+NK-92 cells.IL-15 group:K562 cells expressing IL-15 and 4-1BBL+NK-92 cells.IL-21 group:K562 cells expressing IL-21 and 4-1BBL+NK-92 cells.IL-15+IL-21 group:K562 cells expressing IL-15 and 4-1BBL,K562 cells expressing IL-21 and 4-1BBL+NK-92 cells).Cytotoxicity of NK-92 cells after in vitro amplification was measured by CCK-8 and lactate dehydrogenase release method.To evaluate the anti-tumor effect of NK-92 cells,the level of IFN-γin the supernatant of NK-92 cells was measured by ELISA.Results:Two groups of transfected HEK293FT cells and K562 cells infected with lentiviral solution carried green fluorescence,and the two groups of feeder cells successfully expressed the target gene.The feeder cells in each group could stimulate a large amount of NK-92 cells to proliferate.With the increase of effect to target ratio,the cytotoxicity of NK-92 after expansion was higher in the IL-15 group than in the other groups(P<0.05).When the target ratio was 5∶1 and 10∶1,the difference of IFN-γsecretion by NK cells between IL-15 group and other groups was statistically significant(P<0.05).Conclusion:IL-15 and IL-21 were successfully expressed on the surface of K562 cells in combination with 4-1BBL,respectively,and the effective combination of NK-92 cells with high cytotoxic activity and effective anti-tumor activity in vitro was compared and explored,namely,the IL-15 group may be better used for in vitro expansion of NK-92.

关 键 词：NK-92细胞 白细胞介素-15 白细胞介素-21 4-1BB配体 饲养层 

分 类 号：R730.51[医药卫生—肿瘤]