采用多功能流式细胞术分析分选造血干细胞和髓系定向分化祖细胞

Sorting and analysis of hematopoietic stem cells and myeloid lineage-committed progenitors using flow cytometry

目的探讨富集纯化造血干细胞（HSC）和髓系定向分化祖细胞的新实验方案。方法根据造血干细胞和定向分化祖细胞在发育过程中表达某些特异性分化抗原的特性，通过免疫磁珠分选技术结合四色和六色流式细胞术分析14只健康小鼠的骨髓造血干细胞、造血祖细胞及定向分化祖细胞系列的表达，并对其进行分选，以进一步通过集落细胞培养和传代试验对分选后细胞的活性进行检测。结果经上述实验方案分析，14只健康小鼠骨髓造血祖细胞（HPC）的表达率约为HSC的10倍；但其生成活性远不如造血干细胞，共同髓系祖细胞（CMP）的传代能力仅为HSC的1／2，且次级分化的粒系单核系祖细胞（GMP）和红系巨核系祖细胞（MEP）的生成活性更弱，其传代次数为零。结论通过多色流式细胞术实验方案可以分析纯化HSC和髓系定向分化祖细胞的表达，并精确计数HSC和祖细胞。

Objective To study the experimental protocol for purification and analysis of hematopoietic stem cells （HSC） and myeloid lineage-committed progenitors. Methods According to differentiation antigen expression pattern on hematopoietic stem cells （HSC） and progenitors during hematopoietic development, HSC and progenitors from bone marrow of 14 healthy mice were analyzed and sorted by magnetic nanoparticles and 4-color or 6-color flow cytometry using multiple antibody panels. Sorted HSC and progenitors were further tested by methylcellulose colony forming unit （CFU） and serial replating assays. Results The expression of hematopoietic progenitor cells （HPC） was 10-fold higher expression than that of HSC. However, replating activity of common myeloid progenitors （CMP） was only half of that of HSC. And there was almost no replating activity observed in granulocyte/macrophage lineage-restricted progenitors （GMP） and megakaryocyte/erythrocyte lineage-restricted progenitors （MEP）. Conclusion Multi -parametric flow cytometry could be used to isolate and count HSC and myeloid lineage-committed progenitors accurately.