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本实验以小鼠胚胎主动脉-性腺-中肾(aorta-gonad-mesonephros,AGM)区、胎肝(fetalliver,FL)以及小鼠骨髓(bonemarrow,BM)为研究对象,研究各阶段造血祖细胞在发育过程中的c-kit的表达变化和部位迁移,以及不同发育阶段、不同造血部位来源c-kit+细胞的生物学特性。用流式细胞术检测小鼠胚胎10.5天(embryonic10.5,E10.5)、E11.5的AGM区,E12.5、E14.5、E16.5、E18的FL以及BM来源的单个核细胞中c-kit+细胞的比例。用磁珠分选法分离E10.5AGM区、E14.5FL以及BM来源c-kit+细胞,贴壁培养法分离小鼠E14.5FL基质细胞。建立体外Transwell共培养体系,比较不同来源的c-kit+细胞增殖、分化、迁移以及集落形成能力。流式细胞术检测发现:相对于BM,E10.5AGM区和E12.5FL中c-kit+细胞比例较高。进行体外培养,AGM区来源c-kit+细胞具有较强的增殖潜能,而FL、BM来源的c-kit+细胞在体外培养中更容易分化;AGM区和FL来源的c-kit+细胞比BM来源的c-kit+细胞在体外培养中具有更强的趋化迁移能力。集落形成能力方面,AGM区来源c-kit+细胞具有较强形成混合集落形成单位(CFU-Mix)的能力;FL、BM来源c-kit+细胞则能形成更多粒单系集落形成单位(CFU-GM)。以上结果表明,和FL、BM来源的c-kit+细胞相比,AGM区来源c-kit+细胞具有更强的体外增殖、多系分化潜能以及趋化迁移能力,在未来可能有着更广阔的应用前景。
In this study, aorta-gonad-mesonephros (AGM), fetalliver (FL) and mouse bone marrow (BM) were used to study the effects of different stages of hematopoietic progenitor cells Changes in c-kit expression and site migration during development, and biological characteristics of c-kit + cells from different hematopoietic sites at different developmental stages. Flow cytometry was used to detect mouse embryos 10.5 days (embryonic10.5, E10.5), E11.5 AGM region, E12.5, E14.5, E16.5, E18 FL and BM-derived mononuclear cells The ratio of c-kit + cells. The E10.5AGM region, E14.5FL and BM-derived c-kit + cells were isolated by magnetic bead sorting and mouse E14.5FL stromal cells were isolated by adherent culture. In vitro Transwell co-culture system was established to compare the proliferation, differentiation, migration and colony-forming ability of c-kit + cells from different sources. Flow cytometry revealed a higher proportion of c-kit + cells in E10.5AGM and E12.5FL relative to BM. In vitro culture, c-kit + cells from AGM region had stronger proliferation potential, while c-kit + cells from FL and BM were more easily differentiated in vitro. AGM region and FL-derived c-kit + c-kit + cells have stronger chemotactic migration ability in vitro. In colony-forming ability, c-kit + cells derived from AGM region had the ability to form CFU-Mix. CF-BM cells derived from c-kit + cells could form more CFU- GM). The above results indicate that the c-kit + cells derived from the AGM region have more in vitro proliferation, multi-lineage differentiation potential and chemotactic migration ability compared with the FL-BM derived c-kit + cells and may have broader application prospect in the future .