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目的:探讨体外培养脐带血单个核细胞定向诱导分化为不同阶段红系祖细胞的动力学变化情况。方法:用0.5%甲基纤维素沉降脐带血红细胞及人淋巴细胞分离液密度梯度离心法得到单个核细胞,在含EPO、SCF、IGF-1等细胞因子的无血清培养体系中诱导其定向分化为红系祖细胞,观察细胞增殖、存活率、细胞集落形成情况,并检测不同阶段细胞红系特异性表面标志CD71和CD235a的表达。结果:随着培养时间的延长,细胞数逐渐增多,14 d细胞可扩增140倍左右,收集诱导后的细胞进行瑞氏吉姆萨染色,可见大量红系祖细胞,诱导后的细胞集落形成能力强,形成的克隆大部分为红系集落。诱导过程中,14 d前CD71、CD235a的表达逐渐增高。按细胞表面标志表达的不同可将诱导的细胞分为4群,分别对应红系祖细胞的不同阶段;随着诱导天数的增加,各时间点细胞对应的早期红系祖细胞群(P2、P3)比例逐渐下降,中晚期红系祖细胞群(P4、P5)的比例逐渐上升。结论:无血清培养基添加细胞因子组合的红系诱导培养体系可较好地诱导扩增红系祖细胞,流式分选可获得相对均一而处于不同分化阶段的红系祖细胞群体。获得了红系祖细胞体外分化的动力学数据,为今后进一步优化红系诱导分化体系获得均一的红系祖细胞奠定了基础,并对未来利用干细胞制备均一的红系祖细胞应用于临床治疗有一定的指导作用。
OBJECTIVE: To investigate the kinetic changes of cord blood mononuclear cells induced in vitro by differentiation into erythroid progenitor cells in different stages. Methods: Mononuclear cells were obtained by density gradient centrifugation with 0.5% methylcellulose sedimentation cord blood erythrocytes and human lymphocyte separation fluid, and induced their differentiation in serum-free culture system containing EPO, SCF, IGF-1 and other cytokines As erythroid progenitor cells. The proliferation, survival rate and colony formation of cells were observed, and the expressions of erythroid specific surface markers CD71 and CD235a at different stages were detected. Results: With the prolongation of culture time, the number of cells increased gradually, and the number of cells increased about 140 times after 14 days. The induced cells were collected and stained with Wright Giemsa staining, showing a large number of erythroid progenitor cells, induced colony forming ability Strong, most of the clones formed for erythroid colonies. During induction, the expression of CD71 and CD235a gradually increased before 14 d. According to the different expression of cell surface markers, the induced cells were divided into 4 groups, which corresponded to the different stages of erythroid progenitor cells. With the increase of induction days, the corresponding erythroid progenitor cells (P2, P3 ) Gradually decreased, the ratio of middle and late erythroid progenitor cells (P4, P5) gradually increased. CONCLUSION: Erythroid-derived progenitor cells cultured in serum-free medium supplemented with cytokines can induce the proliferation of erythroid progenitor cells. Flow cytometry can obtain a relatively homogeneous population of erythroid progenitor cells at different stages of differentiation. Obtained kinetic data of in vitro differentiation of erythroid progenitor cells, which laid the foundation for further optimization of erythroid-induced differentiation system to obtain uniform erythroid progenitor cells in the future and the application of stem cell-derived erythroid progenitor cells for clinical treatment in the future A certain guiding role.