单个人造血干细胞体外培养体系用于小分子化合物筛选

Ex vivo Culture System of Single Human Hematopoietic Stem Cell Used to Screan the Small Molecular Compounds

目的:探索一种高效且稳定验证小分子化合物对人造血干细胞(hHSC)调控作用的体系和方法。方法:用流式细胞术结合目前对h HSC表面标记的研究,优化h HSC分选过程,将干细胞培养精确至单个细胞水平,进而研究小分子化合物对细胞干性的影响。用已发表的对人HSC有扩增作用的小分子化合物SR1及UM171处理单个h HSC,14 d后进行细胞表型流式细胞术检测以及细胞涂片观察形态,同时利用CFC和CAFC验证培养后细胞造血功能的变化。结果:多细胞培养中2种小分子化合物及其组合作用与已知研究相符,并由此获得化合物使用浓度。多参数单细胞分选(CD34+CD38-CD45RA-CD90+CD49f+)及体外培养结果与多细胞培养结果一致,细胞形态分析结果与流式细胞术检测结果一致。此外,CFC和CAFC体外造血功能实验中,处理组细胞集落形成能力显著高于对照组(P<0.05)。结论:建立了一个周期短且扩增作用稳定的单个hHSC体外培养体系和验证小分子化合物作用的方法,为小分子化合物筛选建立了可靠的平台并为以后的h HSC扩增研究打下基础。

Objective: To explore an efficient,stable system and method to verify the regulation effect of small molecule compounds on human hematopoietic stem cells( h HSC). Methods: By using combination of flow cytometry w ith study results of surface markers on h HSC,and optimation of sorting process for further studying the effect of small molecular compounds on stem property of h HSC, the single h HSC w as treated w ith published small molecular compounds such as SR1 and UM171 w hich possess the expansion effect. After treating w ith h HSC for 14 d,the flow cytometric analysis of cell phenotypes and cell morphologic observation w ere performed, at the same time the hematopoietic function of cultured h HSC w as verified by colony-forming cell( CFC) test and cobblestone area forming cell( CAFC) test. Results: The effects of SR1 and UM171 and their compositions in multi-cell culture w ere consistent w ith the published data,therefore the useful concentration of compounds w ere obtained. The results of multiparameter sorting of single cell( CD34+CD38-CD45RA-CD90+CD49f+) and ex vivo culture were consistent with the results of bulk cell culture. The results of cell phenotype analysis w as in accordance w ith flow cytometric results. In addition,CFC test and CAFC test revealed that the colony-forming ability in treated group w as significantly higher than that in control group( P < 0. 05). Conclusion: The rapid,efficient stably amplified and short-time culture system for single h HSC and method for varifying the effect of small molecular compounds are established,w hich provides platform for screening small molecular compounds and lays the foundation for further study of h HSC expansion.