脐血CD34+细胞及红系祖细胞扩增的实验研究

脐血是造血祖细胞的丰富来源之一,选择合适的培养条件,体外诱导其定向扩增为红系祖细胞,输入体内产生成熟红细胞。本实验旨在探讨脐血单个核细胞(MNC)体外红系定向扩增的理想因子组合(Flt3配基FL联合TPO、SCF、EPO及FL、SCF、TPO)对CD34 细胞扩增的影响。将单个核细胞接种至stemspan无血清培养液中,共分3组:A组为对照组,B组为TPO SCF FL EPO IGF1组,C组为TPO SCF FL组,C组在第6天及以后换液加入EPO和IGF1。于培养0、6、10、14天进行细胞计数,细胞集落测定,流式细胞术测定细胞的CD34、CD34CD71、CD71GPA细胞的比例。结果表明:经10天培养后,B组总细胞数扩增6.89倍,而C组3.06倍;B组CD34 细胞增加4.83,而C组2.47倍;B组集落形成细胞数增加4.3倍,而C组增加2.5倍;B组红系祖细胞BFUE和CFUE数增加5.4倍,而C组3.1倍;B组CD34 CD71 细胞数增加8.72倍,而C组3.37倍;B组CD71 GPA 细胞数增加53.4倍,而C组30.29倍。结论:脐血MNC在无血清培养液中加入FL SCF TPO实现了CD34 细胞及集落形成细胞的扩增。脐血MNC在无血清培养液中加入FL SCF TPO EPO IGF1短期液体培养获得红系祖细胞的扩增,在第0天比6天加入EPO获得更多红祖细胞(P<0.05)。由于TPO SCF FL EPO IGF1组的集落形成细胞数、CFUE和BFUE数于第10天最多,故培养后收获时

Expansion of Erythroid Progenitors and CD34 + Cells by Umbilical Cord Blood Mononuclear Cells

Cord blood represents a large source of hematopoietic stem/progenitor cells. It can be induced to proliferate directly into erythroid progenitors in a appropriate ex vivo culture condition, then to generate mature red blood cells after injection into the body. The combination of Flt3 ligand,TPO,SCF and EPO is ideal for cord blood MNC to proliferate into erythroid progenitors. This study was aimed to evaluate the effect of FL, SCF and TPO on CD34 expansion. and to investigate influence of the cytokine combination on the proliferation of the CD34 cells . Mononuclear cells (MNC) were cultured in serum-free liquid culture system. Experiments were divided into 3 groups. In the group A as control no cytokines were added in the culture system;in the group B the cells cultured with SCF FL TPO EPO IGF-1; in the group C the cells were cultured with SCF FL TPO. EPO and IGF-1 were added at day 6. Part of the renewed MNCs and colony-forming units were counted, the proportion of CD34 , CD34 CD71 , CD71 GPA cells was detected by FACS. The result showed that after 10 days, the the total cord blood cells increased 6.89-folds in group B and 3.06-folds in group C (P<0.05). The CD34 cells increased 4.83-folds in group B and 2.47-folds in group C (P<0.05). The colony-forming cells (CFCs) increased 4.3-folds in group B and 2.5-folds in group C (P<0.05) . Erythroid progenitors( BFU-E) and CFU-E increased 5.4-folds in group B and 3.1-folds in group C (P<0.05). The CD34 CD71 cells increased 8.72-folds in group B and 3.37-folds in group C (P<0.05). The CD71 GPA cells increased 53.4-folds in group B and 30.29-folds in group C.They were different at any time point (P<0.05). It is concluded that in the group with FL SCF TPO, CD34 cells and CFC can greatly be expanded from cord blood MNC in the serum-free culture system. In the group with FL SCF TPO EPO IGF-1, erythroid progenitors can greatly be expanded in the serum-free culture system, supplying EPO at day 0 was better than supplying at day 6 . Since the largest number of colony-forming cells suchas BFU-E and CFU-E were gained in the TPO SCF FL EPO IGF-1 group at day 10, the harvest time after cultivation in vitro should be selected at day 10.