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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 a system and method for efficiently and stably verifying the regulatory effect of small molecule compounds on human hematopoietic stem cells (hHSCs). METHODS: Flow cytometry was used in combination with the current study of h HSC surface markers to optimize h HSC sorting and to culture stem cells to a single cell level to investigate the effect of small molecule compounds on cell dryness. A single h HSC was treated with the published small molecule compounds SR1 and UM171 that had an amplification effect on human HSC. After 14 days, the cells were detected by flow cytometry and morphological observation by cell smear. Meanwhile, CFC and CAFC were used to verify the culture condition Changes in cell hematopoietic function. Results: The two small molecule compounds in multicellular cultures and their combination are consistent with the known studies and the concentration of the compounds used is thus obtained. Multi-parameter single cell sorting (CD34 + CD38-CD45RA-CD90 + CD49f +) and in vitro culture results were consistent with those of multicellular cultures. The results of cell morphology analysis were consistent with those of flow cytometry. In addition, CFC and CAFC in vitro hematopoietic function experiments, the treatment group colony-forming ability was significantly higher than the control group (P <0.05). CONCLUSION: A single hHSC in vitro culture system and a method of verifying the effect of small molecule compounds with a short cycle and stable amplification activity are established. The establishment of a reliable platform for the screening of small molecule compounds will lay a foundation for the future study of h HSC amplification.