甲基纤维素沉降对脐带血单个核细胞分离效果的影响  

Effect of methyl cellulose sedimentation on the isolation of mononuclear cells from umbilical cord blood

作　　者：袁茵[1] 鲁欣[2] 陈丹亮[3] 邵红伟[1] 黄树林[1] 

机构地区：[1]广东药学院生命科学与生物制药学院/广东省生物技术候选药物研究重点实验室,广东广州510006 [2]华南师范大学生命科学学院,广东广州510631 [3]暨南大学附属第一医院妇产科,广东广州510630

出　　处：《广东药学院学报》2014年第2期216-219,共4页Academic Journal of Guangdong College of Pharmacy

基　　金：广东省医学科研基金(B2013197);国家"重大新药创制"科技重大专项(2009ZX09103-708);国家自然科学基金(31100664;31300737;81303292)

摘　　要：目的研究甲基纤维素沉降预处理对密度梯度离心法分离脐带血单个核细胞效果的影响。方法取等量的同份脐带血,分别采用Ficoll密度梯度离心一步法和甲基纤维素-Ficoll两步法分离脐带血单个核细胞,倒置显微镜下计数并结合台盼蓝拒染法判定细胞活力,流式细胞术检测其中的造血干细胞以及NK细胞亚群的含量。结果通过2种分离方法分离得到的脐带血单个核细胞的数量、细胞活力和CD34+造血干细胞的含量差异无统计学意义,但通过两步法获得的脐带血CD56+、CD3-CD56dim以及CD3-CD56bright等NK细胞亚群的含量比一步法有明显提高。结论甲基纤维素沉降不影响脐带血单个核细胞的得率、细胞活力以及其中的造血干细胞含量,且较好地保存了脐带血中以CD56分子为主要标志的NK细胞亚群。Objective To investigate the effect of methyl cellulose sedimentation on the isolation efficiency of mononuclear cells （MNCs） from umbilical cord blood （UCB） by density gradient centrifugation.Methods Two umbilical cord blood samples with equal volume from the same donor were isolated by Ficoll one-step isolation and methyl eellulose-Fieoll two-step isolation, respectively. The total amount of MNCs was calculated by direct ceil counting under inverted microscope, and the cell viability was assayed by trypan blue negative counting.Furthermore,the percentages of hematopoietic stem cells （HSCs） and natural killer （NK） cells in the UCB MNCs were determined by flow cytometry. Results There was no significant difference in either MNC separating efficiency or cell viability between Ficoll one-step isolation andmethylcellulose-Ficoll two-step isolation.Compared with one-step isolation, the two-step method achieved a similar number of CD34^＋ HSCs, while a higher percentage of CD56^＋, CD3-CD56^dim and CD3-CD56^bright NK cell subpopulations in the UCB MNCs. Conclusion Methylcellulose sedimentation has no influence on the yield, cell activity and HSC frequency of MNCs from UCB isolated by density gradient centrifugation method, and it is helpful in keeping CD56^＋ NK cell subpopulations in the umbilical cord blood.

关 键 词：脐带血 单个核细胞 甲基纤维素 自然杀伤细胞 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]