体外高效活化扩增人脐带血来源自然杀伤细胞的两种体系的比较研究  

Comparative Study of the Two High-Efficient Strategies for in vitro Generation of Human Umbilical Cord Blood-derived Natural Killer Cells

作　　者：王媞尔 孙运艳[1,2] 韩忠朝 张磊升 史明霞 WANG Ti-Er;SUN Yun-Yan;HAN Zhong-Chao;ZHANG Lei-Sheng;SHI Ming-Xia(Department of Hematology,The First Affiliated Hospital of Kunming Medical University,Hematology Research Center of Yunnan Province,Kunming 650032,Yunnan Province,China;Department of Hematology,Yunnan Cancer Hospital,The Third Affiliated Hospital of Kunming Medical University,Yunnan Cancer Center,Kunming 650118,Yunnan Province,China;Key Laboratory of Gastrointestinal Tumor Diagnosis and Therapy of National Health Commission&Key Laboratory of Molecular Diagnostics and Precision Medicine for Surgical Oncology in Gansu Province,Gansu Provincial Hospital,Lanzhou 730000,Gansu Province,China;Key Laboratory of Radiation Technology and Biophysics,Hefei Institute of Physical Science,Chinese Academy of Sciences,Hefei 230031,Anhui Province,China)

机构地区：[1]昆明医科大学第一附属医院血液科,云南省血液病研究中心,云南昆明650032 [2]云南省肿瘤医院(昆明医科大学第三附属医院)血液科,云南省癌症中心,云南昆明650118 [3]国家卫生健康委胃肠肿瘤诊治重点实验室&甘肃省外科肿瘤分子诊断与精准治疗重点实验室,甘肃省人民医院,甘肃兰州730000 [4]辐射技术与生物物理重点实验室,中国科学院合肥物质科学研究院,安徽合肥230031

出　　处：《中国实验血液学杂志》2023年第2期553-561,共9页Journal of Experimental Hematology

基　　金：国家自然科学基金资助项目(No.81760035);中国博士后自然科学基金面上项目(2019M661033);河北省自然科学基金(H2020206403);云南省科技厅-昆明医科大学应用基础研究联合专项基金项目(No.2017FE468-030);贵州省科技计划项目一般项目(黔科合基础-ZK[2021]一般107);中国医学科学院中央级公益性科研院所基本科研业务费专项资金资助(2019PT320005)。

摘　　要：目的:探讨人脐带血来源的单个核细胞(hUC-MNC),经两种体外培养体系活化扩增后的人脐带血自然杀伤细胞(hUC-NK)在生物学表型和细胞毒性上的异同。方法:收集健康供者捐赠的脐带血,经淋巴细胞分离液介导的密度梯度离心法富集单个核细胞。基于本课题组近期建立的3因子体系(定义为“3IL”),比较Miltenyi和XVIVO 15两种培养基体外培养14 d后NK细胞(分别定义为M-NK和X-NK)的细胞表型、细胞亚群、细胞活力及细胞毒功能的相似性和差异性。结果:经上述2种体系诱导14 d后,总CD3^(-)CD56^(+)NK细胞比例由4.25%±0.04%(d 0)提高至71%±0.18%(M-NK)和75.2%±1.1%(X-NK)。与X-NK组相比,M-NK组CD3^(+)CD4^(+)T和CD3^(+)CD56^(+)NKT细胞比例显著降低。X-NK组中表达CD16、NKG2D、NKp44、CD25活化型标志物的细胞比例更高,但M-NK组总NK细胞扩增数量为X-NK组的2倍。两组在NK细胞增殖及细胞周期上无统计学差异,M-NK组的Annexin V^(+)凋亡细胞比例低于X-NK组。NK细胞与K562细胞体外共培养杀伤实验显示,在相同效靶比(E∶T)下,M-NK组CD107a^(+)NK细胞的比例更高,杀伤效率亦略优于X-NK组(P<0.05)。结论:两种体外扩增活化体系均可诱导产生高活化水平的hUC-NK,但二者在生物学表型和肿瘤杀伤毒性等多个方面存在差异。Objective:To explore the similarities and variations of biological phenotype and cytotoxicity of human umbilical cord blood natural killer cells(hUC-NK)after human umbilical cord blood-derived mononuclear cells(hUCMNC)activated and expanded by two in vitro high-efficient strategies.Methods:Umbilical cord blood mononuclear cells(MNC)from healthy donor were enriched by Ficoll-based density gradient centrifugation.Then,the phenotype,subpopulations,cell viability and cytotoxicity of NK cells derived from Miltenyi medium(denoted as M-NK)and XVIVO 15(denoted as X-NK)were compared using a"3IL"strategy.Results:After a 14-day’s culture,the contents of CD3^(-)CD56^(+)NK cells were elevated from 4.25%±0.04%(d 0)to 71%±0.18%(M-NK)and 75.2%±1.1%(XNK)respectively.Compared with X-NK group,the proportion of CD3^(+)CD4^(+)T cells and CD3^(+)CD56^(+)NKT cells in M-NK group decreased significantly.The percentages of CD16^(+),NKG2D^(+),NKp44^(+),CD25^(+)NK cells in X-NK group was higher than those in the M-NK group,while the total number of expanded NK cells in X-NK group was half of that in M-NK group.There were no significant differences between X-NK and M-NK groups in cell proliferation and cell cycle,except for the lower percentage of Annexin V^(+)apoptotic cells in M-NK group.Compared with X-NK group,the proportion of CD107a^(+)NK cells in M-NK group were higher under the same effector-target ratio(E∶T)(P<0.05).Conclusion:The two strategies were adequate for high-efficient generation of NK cells with high level of activation in vitro,however,there are differences in biological phenotypes and tumor cytotoxicity.

关 键 词：NK细胞 活化扩增 细胞亚群 细胞活力 细胞毒性 

分 类 号：R392[医药卫生—免疫学]