三种不同方法分离脐血造血干细胞的效果比较  

Comparison of 3 Different Methods on Isolation of Umbilical Cord Blood Hematopoietic Stem Cells

作　　者：王颖超[1] 殷楚云[1] 冯磊[1] 赵小明[1] 王晓晖[2] 徐彦磊[3] 

机构地区：[1]郑州大学第一附属医院儿科,郑州450052 [2]郑州大学第一附属医院妇产科,郑州450052 [3]郑州大学第一附属医院小儿外科,郑州450052

出　　处：《实用儿科临床杂志》2012年第10期766-768,794,共4页Journal of Applied Clinical Pediatrics

摘　　要：目的探讨一种较好的脐血造血干细胞(HSCs)的分离方法,以最大限度地分离出脐血中的HSCs,使其以高纯度、最佳生长状态用于治疗和研究。方法取本院足月妊娠健康产妇的脐血20份,每份均采用羟乙基淀粉沉淀(HES)法、淋巴细胞分离液(Ficoll)密度梯度离心法、免疫磁珠(MACS)法处理。比较3种方法分离后的单个核细胞(MNCs)回收率、锥虫蓝拒染率及CD34细胞阳性率;将以上3种方法所得细胞用完全培养基培养,不同时间点计数细胞,绘制生长曲线,并观察原代细胞的生长情况及其形态特征;并采用半固体琼脂培养法,计数其粒-巨噬细胞集落形成单位数。结果 HES法得到的MNCs回收率高于Ficoll密度梯度离心法和MACS法(Pa<0.05);锥虫蓝拒染率三者比较差异无统计学意义(P>0.05);MACS法所得细胞CD34阳性率明显高于另2种方法(Pa<0.05),并且其所得细胞生长状态好,集落数最多;HES法和Ficoll法所得细胞,部分呈贴壁生长,与贴壁细胞共培养的细胞生长状态好;Ficoll密度梯度离心法得到细胞生长状态最差,集落数最少。结论 HES法可作为一种首选常规分离脐血造血干细胞的方法;MACS法分离得到造血干细胞纯度高,适于实验及临床研究。Objective To explore a better method to isolate hematopoietic stem cells（HSCs） from umbilical cord blood and make the HSCs used for treatment and research with the high purity and best growth state. Methods Twenty umbilical cord blood samples were collected and separated using hydroxyethyl starch （HES） method, Ficoll densify gradient centrifugation method and immunomagnetic beads （MACS） method, respectively. The rate of recollection for the mononuclear cells（MNCs） , trypan blue dye viability and CD~ cells were compared a- mong the 3 methods. The number of MNCs at different times, growth conditions, morphologic changes of primary cells and colony - forming u- nit - granuloeyte macrophage were detected. Results The rate of recollection for MNCs using HES method was higher than that of using Fi-coll method and MACS method （Pa 〈 0.05 ）. The rate of refusing trypan blue among the 3 methods was no difference （P 〉 0.05）. The rate of CD34 cells isolated by MACS was higher than that of the other two methods （Pa 〈 0.05 ） , and the MNCs were kept in a good condition. There were adherent cells in the MNCs isolated by HES and Ficoll method at the culture medium and these adherent cells could help the growth of MNCs. The MNCs isolated by Fieoll method were kept in a bad condition. Conclusions HES method is an appropriate method for separating HSCs. MACS method is better for further experimental and clinical research.

关 键 词：脐血 造血干细胞 分离 间充质干细胞 

分 类 号：R457[医药卫生—治疗学]