免疫磁珠法分选人骨髓多能成体祖细胞及初步鉴定

In vitro purification and culture of multipotent adult progenitor cells from human bone marrow by magnetic activated cell sorting

目的：应用免疫磁珠法分选人骨髓多能成体祖细胞,观察其分选效果,建立体外分选纯化及培养人骨髓来源多能成体祖细胞（hMAPCs）的方法.方法：取健康成人志愿者适量骨髓后采用梯度密度离心法分离获取单个核细胞,在自制培养基下贴壁培养后,将获取的骨髓贴壁细胞通过CD45、血糖蛋白A（GlyA）免疫微磁珠（miniMACS）负分选,锥虫蓝拒染实验计数MACS分选前后细胞活力.流式细胞仪鉴定分选后细胞纯度;流式细胞仪分析培养细胞CD29、CD44、CD34和HLA-DR表达情况.结果：通过MACS分选,平均每1×106/ml骨髓贴壁细胞可分选出约（5～10）×104/ml hMAPCs,分选后的hMAPCs细胞生长良好,最长传代到第20代.分选前后细胞活力分别为（96.7±1.7）%和（96.0±2.4）%,无明显差异;流式细胞仪分析获取的CD45-、GlyA-细胞纯度大于98%;流式细胞仪检测hMAPCs中CD29阳性表达细胞比率为99.2%,CD44阳性细胞比率为98.3%,CD34阳性细胞比率为1.2%,HLA-DR阳性细胞比率为5.3%.结论：CD45、GlyA免疫微磁珠负分选可从骨髓中分离高纯度的hMAPCs;分选后hMAPCs在自行研制的培养基中有较强的增殖能力.

Objective：To establish a method for isolation, purification, and culture of human multipotent adult progenitor cells （MAPCs） in vitro, so as to lay a foundation for the application of hMAPCs in tissue-engineering research and clinical medicine. Methods： The mononuclear cells were obtained from bone marrow of healthy volunteers by gradient centrifugation and were subjected to adherence culture. The adherent cells were then subjected to magnetic activated cell sorting （MACS） （deple tion selection with CD45 and GlyA microbeads）. Then the purity of selected cells was identified by flow cytometer. The growth of the purified cells was observed and the expression of CD29, CD44, CD34, and HLA-DR was analyzed by flow cytometers. Results ： （1） Averagely （5-10）×10^4/ml hMAPCs could be separated from 1×10^6/ml bone marrow mononuclear cells by MACS. The cell viability was similar before （[96.7±1.7]%） and after （[96.0±2.4]%） isolation. （2）The isolated MAPCs grew well in the self-designed culture medium and could be passaged for 20 generation. （3）The purity of the CD45^- and GlyA^- cells separated from bone marrow adherent cells was more than 98 0% as determined by flow cytometer. （4） In hMAPCs, the positive rate was 99.2% for CD29, 98.3% for CD44, 1.2% for CD34, and 5.3% for HLA-DR. Conclusion： （1）The bone marrow-derived hMAPCs can be purified by MACS through depletion selection of CD45 and GIyA microbeads. （2）The hMAPCs can be expanded in vitro in self designed medium, maintaining a non-differentiation state for a long time.