脐血来源NK细胞的体外扩增及其抗肿瘤细胞活性的研究  

Efficient expansion of NK cells derived from cord blood in vitro and their killing activity of tumor cells

作　　者：韩强 张志刚 刘坤 朱贤兑 金媛媛[3] 张志斐 杨兆勇[3] HAN Qiang;ZHANG Zhi-gang;LIU Kun;ZHU Xian-dui;JIN Yuan-yuan;ZHANG Zhi-fei;YANG Zhao-yong(School of Pharmacy,North China University of Science and Technology,Tangshan 063210,China;Department of Pathology,Cangzhou People's Hospital,Hebei 061000,China;Department of Microbial Pathway Engineering,Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing 100050,China)

机构地区：[1]华北理工大学药学院,唐山063210 [2]沧州市人民医院病理科,沧州061000 [3]中国医学科学院北京协和医学院医药生物技术研究所代谢工程室,北京100050

出　　处：《中国医药生物技术》2022年第5期412-417,共6页Chinese Medicinal Biotechnology

基　　金：河北省自然科学基金(B2020209001);国家自然科学基金面上项目(81872782)。

摘　　要：目的研究脐血单个核细胞来源NK细胞的培养方法及其对不同类型肿瘤细胞的杀伤活性。方法自脐静脉消毒后全封闭采集脐血,肝素抗凝,分离单个核细胞,用NK细胞培养基培养14 d,流式细胞仪检测其表面标志,并使用RTCA实时无标记细胞分析技术检测细胞增殖曲线,观察NK效应细胞按效靶比1:1、5:1与10:1对肿瘤细胞的杀伤作用。结果培养14 d的脐血单个核细胞,细胞总数扩增294倍,其中NK细胞扩增5580倍。NK细胞CD3^(-)CD56^(+)阳性率为97.93%,与杀伤功能相关的重要分子CD16、NKG2D、NKp30、NKp44的表达阳性率均在95%以上。对A549肺癌细胞株作用6 h的杀伤活性,在1:1~10:1效靶比范围内,随着效靶比增加,杀伤活性从(78.16±2.41)%逐渐增强到(95.71±3.21)%;并且在效靶比1:1的情况下,随着作用时间从6 h延长到24 h,杀伤活性也从(78.16±2.41)%逐渐增强到(96.33±2.15)%。结论建立了无需分选细胞、无需滋养层细胞、操作简单、通用性强的脐血NK细胞培养方法,获得的NK细胞纯度高,对肿瘤细胞的杀伤作用强。Objective To study the culture method of NK cells derived from cord blood mononuclear cells and their killing activity of different types of tumor cells.Methods After disinfection of umbilical vein,umbilical cord blood was collected completely and anticoagulated with heparin,then the umbilical cord blood mononuclear cells were isolated and cultured in NK cell culture medium for 14 days.Their surface markers were detected by flow cytometry.The cell proliferation curve and the killing effects of NK effector cells on tumor cells were examined according to the effective target ratio of 1:1,5:1 and 10:1 by RTCA real-time unlabeled cell analysis technology.Results Cord blood mononuclear cells cultured for 14 days expanded 294 times in total,including 5580 times in NK cells.The positive rate of CD3-CD56+cell subsets of NK cells cultured for 14 days was 97.93%,and the positive rate of CD16,NKG2D,NKp30 and NKp44 importantly for killing function were more than 95%.When the NK-killing activity of A549 lung cancer cell line for 6 h was in the range of 1:1-10:1 of effector:target ratio,the killing activity gradually increased from(78.16±2.41)%to(95.71±3.21)%with the increase of the ratio;The killing activity increased from(78.16±2.41)%to(96.33±2.15)%with the extension of action time from 6 h to 24 h when the effective target ratio was 1:1.Conclusion This method does not need cell sorting and trophoblast cells.It has the advantages of simple operation,strong universality,high purity of cultured umbilical cord blood NK cells and strong lethality to tumor cells.

关 键 词：自然杀伤细胞 脐血 表面标志 实时无标记动态细胞分析技术 体外扩增 抗肿瘤 

分 类 号：R730.51[医药卫生—肿瘤]