人NK细胞的体外扩增及其基因表达通路的研究  

IN VITRO AMPLIFICATION AND GENE EXPRESSION PATHWAY OF HUMAN NK CELLS

作　　者：张红[1] 吴昊[2] 姜磊 鲁晓晴[1] ZHANG Hong;WU Hao;JIANG Lei;LU Xiaoqing(School of Public Health, Qingdao University, Qingdao 266071, China)

机构地区：[1]青岛大学公共卫生学院,山东青岛266071 [2]青岛大学华赛医学细胞和蛋白质药物研究院

出　　处：《精准医学杂志》2022年第2期170-174,共5页Journal of Precision Medicine

基　　金：国家重点研发计划项目(2018YFA0900802)。

摘　　要：目的通过自然杀伤(NK)细胞体外培养和转录组学测序(RNA-seq)技术研究人NK细胞体外扩增情况及其功能活化的信号通路。方法分离健康志愿者外周血单个核细胞(PBMC),应用NK细胞培养法进行培养。采用流式细胞术、ELISA法和磁性细胞分选技术分析体外培养扩增不同阶段的NK细胞。获得纯化后的NK细胞进行RNA-seq检测,分析人NK细胞体外扩增情况及其功能活化的信号通路。结果体外培养14 d后,NK细胞(CD3-CD56+)的比例达到90%以上。第10、14天NK细胞的杀伤活性均较第0天明显增强,且随着效靶比从5∶1、10∶1到20∶1,各组NK细胞对K562细胞的杀伤活性均逐步提高(F=124.40~5512.00,P<0.05)。培养至第14天的NK细胞IFN-γ的分泌量显著高于第0、10天(q=67.17、7.75,P<0.05)。RNA-seq分析结果显示,第10、14天NK细胞与第0天NK细胞相比差异表达基因(DEGs)均较多,且第10天DEGs多于第14天。GO功能和KEGG通路富集分析显示这些DEGs主要与炎症反应、免疫反应、细胞分裂和增殖相关,而且显著富集在细胞周期、成骨细胞分化、造血细胞调控、NF-Kappa B等信号通路。结论本研究发现CD16抗体联合5种细胞因子的培养方法可以有效诱导PBMC成为NK细胞,为NK细胞免疫治疗提供了理论基础和实验依据。Objective To investigate the in vitro amplification of human natural killer(NK)cells and the signaling pathway of functional activation by in vitro NK cell culture and transcriptome sequencing(RNA-seq)technologies.Methods Peripheral blood mononuclear cells(PBMC)were isolated from the blood of healthy volunteers and cultured using the NK cell culture method.NK cells at different stages of in vitro culture and amplification were analyzed by flow cytometry,ELISA,and magnetic-activated cell sorting techniques.Purified NK cells were obtained for RNA-seq assay to analyze the in vitro amplification of human NK cells and the signaling pathway of functional activation.Results After 14 days of in vitro culture,the proportion of NK cells(CD3-CD56+)reached more than 90%.The killing activity of NK cells was significantly enhanced on days 10 and 14 compared with that on day 0,and the killing activity of NK cells against K562 cells increased gradually with the effector/target ratio from 5∶1,10∶1 to 20∶1 in all groups(F=124.40-5512.00,P<0.05).The amount of interferon-γsecretion of NK cells cultured until day 14 was significantly higher than that on days 0 and 10(q=67.17,7.75,P<0.05).RNA-seq analysis showed that there were more differentially expressed genes(DEGs)in NK cells on days 10 and 14 compared with NK cells on day 0,and there were more DEGs on day 10 than on day 14.The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses showed that these DEGs were mainly associated with inflammatory response,immune response,cell division,and proliferation and significantly enriched in cell cycle,osteoblast differentiation,hematopoietic cell regulation,and NF-Kappa B signaling pathways.Conclusion This study found that the culture method of CD16 antibody combined with five cytokines can effectively induce PBMCs to become NK cells,providing a theoretical basis and experimental evidence for NK cell immunotherapy.

关 键 词：杀伤细胞 天然 K562细胞 干扰素Γ 转录组测序 基因表达谱 免疫疗法 细胞培养技术 

分 类 号：R392.12[医药卫生—免疫学]