体外长期维持中性粒细胞活性的条件培养体系的建立  

Establishment of a Conditioned Culture System to Maintain the Activity of Neutrophils In Vitro for a Long Time

作　　者：王彤 范玉龙 邵雅 赵红菲 刘欢 张诗悦 赵梓含 成天然 许元富 WANG Tong;FAN Yulong;SHAO Ya;ZHAO Hongfei;LIU Huan;ZHANG Shiyue;ZHAO Zihan;CHENG Tianran;XU Yuanfu(State Key Laboratory of Experimental Hematology,National Key Laboratory of Blood and Health,Haihe Laboratory of Cell Ecosystem,Institute of Hematology&Blood Diseases Hospital,Chinese Academy of Medical Sciences&Peking Union Medical College,Tianjin 300020,China;Tianjin Institutes of Health Science,Tianjin 301600,China)

机构地区：[1]中国医学科学院血液病医院(中国医学科学院血液学研究所),血液与健康全国重点实验室,国家血液系统疾病临床医学研究中心,细胞生态海河实验室,天津300020 [2]天津医学健康研究院,天津301600

出　　处：《中国细胞生物学学报》2024年第7期1367-1376,共10页Chinese Journal of Cell Biology

基　　金：中国医学科学院医学科学创新基金(批准号:2021-12M-1-017);国家自然科学基金(批准号:81970107)资助的课题。

摘　　要：中性粒细胞输注是一种临床辅助抗感染治疗方案,而用于输注的中性粒细胞的体外活性维持是影响该方案实施的有效性的关键点,目前也是相关研究的难点。因此,该文探索间充质干细胞(mesenchymal stem cell,MSC)培养上清与一些药物分子联合使用来延缓中性粒细胞的体外衰老和维持机体免疫功能,以期克服上述难题。通过分别采用不同培养条件(包括多种MSC培养上清、酸碱度调节培养基、影响中性粒细胞存活的多种药物分子及其组方等条件培养基)进行中性粒细胞的体外培养,并分别检测中性粒细胞的体外寿命及生物学功能。实验结果显示,相较于对照组(RPMI-1640培养基),MSC培养3天的培养上清可以更好地延长中性粒细胞的体外寿命,同时也可以维持中性粒细胞的ROS释放和吞噬等杀菌功能,两组之间具有显著性差异(P<0.01);通过进一步实验筛选和验证,与单纯使用MSC培养3天的培养上清相比,使用优化后的中性粒细胞条件培养基(MSC培养上清,pH为6.0,1μmol/L nicotine、10 ng/mL G-CSF、0.6 mmol/L NAC、10μmol/L DFP)显著延长了中性粒细胞的体外寿命(P<0.01)。总之,该研究发现间充质干细胞培养上清和多种药物分子组合可以更好地维持中性粒细胞的体外活性,这将为中性粒细胞的体外保存和输注研究提供较好的实验基础和理论依据。Neutrophil transfusion is a clinical adjunctive anti-infective therapy,and the maintenance of in vitro activity of neutrophils used for transfusion is a key point affecting the effectiveness of this therapy,which is currently a challenging aspect of related research.Therefore,this study explored the combined use of MSC-con-ditioned medium and some drug molecules to delay neutrophil in vitro senescence and maintain immune function,to overcome the above difficulties.Neutrophils were cultured in vitro using various conditions(including multiple supernatants of MSC culture,pH regulation of culture medium,various drug molecules and their formulas affecting the survival of neutrophils).The in vitro lifespan and biological functions of neutrophils were measured.The exper-imental results showed that compared with the control group(RPMI-1640 medium),the conditioned medium cul-tured for three days with MSCs could better prolong thein vitro lifespan of neutrophils while maintaining neutrophil func-tions such as ROS release and phagocytosis,with significant differences between the two groups(P<0.01).Further experimental screening and verification revealed that compared with the use of conditioned medium cultured for three days with MSCs alone,the optimized neutrophil conditioning medium(MSC-conditioned medium,pH6.0,1μmol/L nicotine,10 ng/mL G-CSF,0.6 mmol/L NAC,10μmol/L DFP)significantly prolonged the in vitro lifespan of neutrophils(P<0.01).In conclusion,this study found that the combination of mesenchymal stem cell-conditioned medium and multiple drug molecules could better maintain the in vitro activity of neutrophils,providing a good experimental basis and theoretical basis for the in vitro preservation and transfusion research of neutrophils.

关 键 词：中性粒细胞 间充质干细胞 培养上清 小分子药物 凋亡 

分 类 号：R392[医药卫生—免疫学]