羟基淀粉沉淀对免疫磁珠法分离脐血CD_(34)^+细胞及造血祖细胞增殖功能的影响

Colony culture and purification of CD_(34)^+ hematopoietic stem/ progenitor cells from cord blood by MACS and HES

目的 :应用羟基淀粉沉淀去除脐血红细胞和免疫磁珠 (MACS)阳性选择 ,建立富集、分离脐血CD34+ 细胞的简易实用方法 ,通过造血祖细胞集落培养分析其在细胞因子作用下的增殖潜能 ,观察羟基淀粉沉淀对造血细胞增殖功能的影响。方法 :采用羟基淀粉沉淀和改进的 MACS富集、分离脐血 CD34+细胞 ,以甲纤半固体造血细胞培养法观察其粒 -单系细胞 (CFU- GM)和红系爆式集落 (BFU- E)的数量和特性。结果 :脐血分离前 ,CD34+细胞纯度为 1.5 %～ 3% ,MACS分离后其纯度可达 85 % ,羟基淀粉沉淀和 MACS分离可达 91% ;CD34+造血祖细胞培养有明显的增殖 ,采用 IL - 3、IL - 6、SCF、GM- CSF、EPO等细胞因子组合扩增培养 9～ 14d,CD34+ 细胞在液体培养中有明显扩增 ,MACS分离后 CD34+ 细胞总数增加 39倍 ,羟基淀粉沉淀和 MACS分离可增加 45倍。结论 :应用羟基淀粉沉淀和改进的 MACS系统可有效富集、分离脐血 CD34+细胞 ,羟基淀粉沉淀对 CD34+细胞的富集、分离无影响 ;脐血细胞分离后作造血祖细胞培养 ,可产生丰富的 CFU- GM和 BFU- E,说明羟基淀粉沉淀分离的 CD34+ 细胞的增殖功能优于 MACS分离 CD34+ 细胞的增殖功能。

Objective:To explore the methods of cell separation with HES and imunomagnetic beads and its application in the CD 34 + cells methods.To investigate the effect for colony culture and purification of CD 34 + hematopoietic stem/progenitor cells from cord blood by MACS and HES.A new sterilized separation system for purification of CD 34 + hematopoietic stem/progenitor cell was established according to the strategy of positive selection on immunomagnetic separation.Method:Mini Magnetic Cell Sorting(MACS)was applied to select CD 34 + from cord blood (CB).The degree of enrichment was determined by FACS and immunofluorescent method. Meanwhile,colony culture of hematopoietic progenitors was performed to reflect the proliferative potential of the enriched cord blood cells.Result:The percentage of CD 34 + cells was increased from 1.5 %~3% to 91% with a average of viable cells more than 96%.The number of CFU granulocyte macrophage (CFU GM) from CD 34 + cells were (219±62)/2×10 3 and the number of burst forming unit erythroid (BFU E) (84±14)/2×10 3 respectively.Conclusion:The availability of pure population of CD 34 + cells will facilitate further study on biological features and functions of human stem/progenitor cells.HES and MACS has the effectiveness of separation and high viability of cells.