Ficoll分离法富集脐带血自然杀伤细胞的实验条件分析  

Analysis of the Experimental Conditions Affecting the Enrichment of Natural Killer Cells from Umbilical Cord Blood by Ficoll Density Gradient Centrifugation

作　　者：曹倩 冉凤萍 孙海[1,2] 蒙露 陈巧 段曦 郑武燕 刘佳慧 李华 邹强 Cao Qian;Ran Fengping;Sun Hai;Meng Lu;Chen Qiao;Duan Xi;Zheng Wuyan;Liu Jiahui;Li Hua;Zou Qiang(Department of Basic Medical Sciences,Chengdu Medical College,Chengdu 610500,China;Center for Scientific Research,Chengdu Medical College,Chengdu 610500,China;Department of Obstetrics,The Affiliated Hospital of Chengdu Medical College,Chengdu 610500,China;Xi'an Haitang Vocational College,Xi'an 710038,China;Department of Basic Medical Sciences,Chengdu University of T.C.M,Chengdu 610075,China;Department of Oncology,Western Theater General Hospital,Chengdu 610083,China)

机构地区：[1]成都医学院基础医学院,成都610500 [2]成都医学院科研实验中心,成都610500 [3]成都医学院第一附属医院产科,成都610500 [4]西安海棠职业学院,西安710038 [5]成都中医药大学基础医学院,成都610075 [6]西部战区总医院肿瘤科,成都610083

出　　处：《成都医学院学报》2020年第1期6-12,共7页Journal of Chengdu Medical College

基　　金：四川省科技厅应用基础重点项目(No:2018JY0440);四川省科技厅应用基础重点项目(No:19YYJC0242)

摘　　要：目的探讨影响Ficoll分离法富集脐带血(UCB)自然杀伤细胞(NK)的实验条件。方法在不同保存温度、复温时间、Ficoll密度和抗凝剂比例条件下,Ficoll法分离临床UCB,通过流式检测NK存活率、富集前后的回收率以及粒细胞清除率,评判富集效果。结果UCB在4、25、37℃下可保存至7h,随后UCB中NK存活率和富集回收率、粒细胞清除率随时间延长明显下降。对长时间保存的4℃脐血,可在不影响NK存活率情况下,通过复温37℃1h或适度增加Ficoll密度提升回收率。此外,血袋规格应与采集血量相匹配,否则较少血量会导致预留抗凝剂在UCB中的浓度变大,也会降低NK富集回收率。结论在保持UCB与抗凝剂正常比例情况下,临床UCB 37℃保存7h内进行Ficoll分离法富集NK可达到较好富集效果。Objective To investigate the experimental conditions affecting the enrichment of nature killer(NK)cells from umbilical cord blood(UCB)by Ficoll density gradient centrifugation.Methods UCB was clinically separated by Ficoll density gradient centrifugation under the conditions of different storage temperature,rewarming time,Ficoll density and anticoagulant ratio.The survival rate of NK cells,recovery rate before and after enrichment and clearance rate of granulocytes were measured by flow cytometry to evaluate the enrichment effect.Results UCB could be preserved for 7 hours at 4℃,25℃and 37℃respectively,and the survival rate,enrichment recovery rate and granulocyte clearance rate of NK cells in UCB decreased with time after 7 hours.The recovery rate of UCB preserved at 4℃for a long time could be improved by rewarming UCB to 37℃or moderately increasing the Ficoll density without affecting the survival rate of NK cells.The size of blood bags should match the collected blood volume,or the less blood volume would lead to the increase of the anticoagulant concentration in UCB and the decrease of the enrichment recovery rate in NK cells.Conclusion The better enrichment effect of NK cells from UCB can be achieved by Ficoll density gradient centrifugation after the clinical UCB is preserved at 37℃for 7 hours under the condition of keeping the normal ratio of UCB and anticoagulant.

关 键 词：脐血 NK细胞 Ficoll分离法 回收率 

分 类 号：R392.12[医药卫生—免疫学]