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目的 为了探讨脐血CD34~+干/祖细胞在不同细胞因子支持下体外扩增过程中Caspase-3表达及意义,我们采用了RT-PCR、Western blot和流式细胞仪技术测定了脐血CD34~+细胞在体外扩增过程中的生物学特性及Caspase-3的表达。检测结果显示,Caspase-3mRNA在新鲜分离的脐血CD34~+细胞中低水平表达,在细胞因子支持下体外培养3天,扩增的CD34~+细胞中Caspase-3mRNA和蛋白质水平表达上调,但在这两种细胞中仅能检测到分子量为32KDa的非活性酶原形式的Caspase-3;随着体外培养时间的延长,在IL-3,IL-6和GM-CSF组合条件下,Caspase-3被激活,可检测到分子量力20KDa的裂解片段。本研究表明,虽然造血干细胞的凋亡是个复杂的过程,但在脐血CD34~+干/祖细胞体外扩增过程中,Caspase-3参与了凋亡事件并发挥着重要的作用。
Objective To investigate the expression and significance of Caspase-3 during the in vitro expansion of cord blood CD34 ~ + stem / progenitor cells with different cytokines, RT-PCR, Western blot and flow cytometry were used to detect the expression of CD34 Biological characteristics and expression of Caspase-3 in ~ + cells during in vitro expansion. The results showed that Caspase-3 mRNA was expressed at low level in freshly isolated cord blood CD34 + cells and cultured in vitro for 3 days with cytokine support. The expression of Caspase-3 mRNA and protein in CD34 + cells was up-regulated Only 32 kDa non-active zymogen forms of Caspase-3 could be detected in these two kinds of cells. With the prolonging of culture time in vitro, the expression of Caspase-3 in the combination of IL-3, IL-6 and GM- 3 is activated, a 20 kDa fragment of molecular weight can be detected. This study shows that although hematopoietic stem cell apoptosis is a complex process, Caspase-3 participates in the apoptotic event and plays an important role in the in vitro expansion of cord blood CD34 + stem / progenitor cells.