体外诱导骨髓细胞向肥大细胞分化的方法学研究

Study on differentiation of bone marrow cells into mast cells in vitro

目的:探讨体外诱导小鼠骨髓细胞分化为骨髓来源肥大细胞(BMMC)的方案,用于肥大细胞功能调节药物研究。方法:从小鼠股骨取骨髓细胞,在干细胞因子(SCF)、IL-3或SCF和IL-3联合诱导下,体外培养4周。每周用倒置显微镜观察细胞生长情况,统计活细胞总数;流式细胞仪分析培养体系中BMMC比例,并计算BMMC绝对数;4周后通过测定β-己糖胺酶释放量及抑制剂的调节作用评估BMMC功能。结果:3种诱导方法均能促进BMMC体外分化,但不同诱导条件获得的BMMC纯度和数量有较大差异。SCF单独诱导获得的BMMC纯度和数量最低;IL-3单独诱导获得较高的BMMC纯度和总数;SCF和IL-3联合诱导能够获得较高的BMMC纯度,同时能获得远高于两种因子单独诱导获得的BMMC数量;SCF和IL-3联合诱导4周获得的BMMC具有完整功能。结论:SCF和IL-3联合诱导4周能够获得大量高纯度且功能完整的BMMC,可用于探索肥大细胞功能调节药物的活性和作用机制。

Objective:To investigate protocol of inducing differentiation of mouse bone marrow cells into bone marrow-derived mast cell(BMMC)in vitro and to evaluate activities of drugs that can regulate mast cell function.Methods:Bone marrow cells were prepared from mouse femurs and cultured in vitro for 4 weeks under induction of stem cell factor(SCF),IL-3 or combination of SCF and IL-3.Cell growth was observed and total live cells were counted with an inverted microscope every week.BMMC percentage was analyzed by flow cytometry,and absolute number of BMMC was calculated.After 4 weeks,BMMC function was evaluated by measuring release ofβ-hexosaminidase and regulatory effect of inhibitor.Results:All three induction methods could promote differentiation of BMMC in vitro,while purity and quantity of BMMC obtained under different induction conditions were different.Induction with SCF got the lowest purity and number of BMMC.Treatment with IL-3 significantly increased purity and number of BMMC.Combination of SCF and IL-3 significantly improved purity of BMMC and acquired maximum quantity of BMMC.BMMC obtained after 4 weeks of combined SCF and IL-3 induction was fully functional.Conclusion:SCF and IL-3 combined induction for 4 weeks can obtain a large number of high purity and functional BMMC,which can be used to explore activities and mechanisms of mast cell function-modulating drugs.