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本研究探讨DAPT(N-[N-(3,5-difluorophenacetyl-L:-alanyl)]-S-phenylglycinet-butyl ester)对小鼠骨髓造血干细胞(HSC)细胞周期、凋亡、分化及扩增的影响及其可能的相关机制。运用实时定量PCR检测DAPT1μmol/L作用前后细胞周期相关基因p18、p21、p27、CDK1、CDK2、CDK4、CDK6 mRNA表达水平及凋亡相关基因Bcl-2、Bcl-xl、mcl-1、Bax、Bim、p53、Puma mRNA表达量的水平;用流式细胞术检测DAPT作用前后小鼠骨髓细胞Lin-c-kit+Sca-1+及CD34-Lin-c-kit+Sca-1+表型细胞的细胞周期及凋亡的变化;用单细胞培养检测DAPT作用前后单个HSC分化的变化;用长周期培养实验检测DAPT作用前后HSC扩增能力的变化。结果显示,Lin-c-kit+Sca-1+表型细胞经DAPT处理5 d后,小鼠骨髓细胞中细胞周期相关基因CDK1、CDK2、CDK4、CDK6及p27的mRNA表达量较对照组明显升高(P<0.01-P<0.001),p18和p21表达量明显降低(P<0.01-P<0.001);凋亡相关基因Bcl-2、Bcl-xl、Bax、p53、Puma表达量较对照组明显升高(P<0.01-P<0.001),Bim表达量明显降低(P<0.001),Mcl-1的表达量无差异。加DAPT处理5 d后,小鼠骨髓细胞Lin-c-kit+Sca-1+表型细胞的细胞周期的变化无统计学意义(P>0.05),小鼠骨髓细胞CD34-Lin-c-kit+Sca-1+表型细胞的G0期的细胞减少,G1期的细胞明显增多(P<0.05),处于S、G2、M期的细胞数量的变化无统计学意义(P>0.05);小鼠骨髓细胞Lin-c-kit+Sca-1+表型细胞和CD34-Lin-c-kit+Sca-1+表型细胞的细胞凋亡增多(P<0.05)。单细胞培养10 d实验显示,每板形成的克隆数、每孔平均细胞数的变化及DAPT对单个造血干细胞分化影响的差异均无统计学意义(P>0.05)。DAPT处理3 d后,小鼠骨髓细胞中HSC的扩增能力下降。结论:DAPT可加速小鼠骨髓细胞CD34-Lin-c-kit+Sca-1+表型细胞的耗竭,促进小鼠骨髓细胞Lin-c-kit+Sca-1+及CD34-Lin-c-kit+Sca-1+表型细胞的凋亡,降低小鼠骨髓细胞中HSC的扩增能力,但对单个CD34-Lin-c-kit+Sca-1+表型细胞的增殖及分化无明显影响。
This study was aimed to investigate the cell cycle, apoptosis, differentiation and proliferation of mouse bone marrow hematopoietic stem cells (HSCs) induced by DAPT (N- (3,5-difluorophenacetyl-L: -alanyl) The impact and possible related mechanisms. The expression of p18, p21, p27, CDK1, CDK2, CDK4 and CDK6 mRNA and the expression of Bcl-2, Bcl-xl, Bcl-xl, Bax and Bim were detected by real-time PCR before and after DAPT1μmol / , P53 and Puma mRNA levels were detected by flow cytometry. The expression of Lin-c-kit + Sca-1 + and CD34-Lin-c-kit + Sca-1 + cells in bone marrow of mice before and after DAPT treatment Cell cycle and apoptosis. The single-cell culture was used to detect the differentiation of single HSCs before and after DAPT treatment. The long-period culture experiments were performed to detect the changes of HSCs’ ability to proliferate before and after DAPT treatment. The results showed that the mRNA expression of CDK1, CDK2, CDK4, CDK6 and p27 in bone marrow cells of Lin-c-kit + Sca-1 + -treated cells was significantly increased compared with that of the control group The expression of Bcl-2, Bcl-xl, Bax, p53 and Puma in the control group was significantly higher than that in the control group (P <0.01-P <0.001) (P <0.01-P <0.001), the expression of Bim was significantly lower (P <0.001), and the expression of Mcl-1 was no difference. After 5 days of DAPT treatment, there was no significant difference in cell cycle between Lin-c-kit + Sca-1 + phenotype cells in mouse bone marrow cells (P> 0.05). CD34-Lin-c-kit The number of cells in G0 + Sca-1 + phenotype cells was decreased, the number of cells in G1 phase was significantly increased (P <0.05), and the number of cells in S, G2 and M phases was not significantly changed (P> 0.05) The apoptosis of Lin-c-kit + Sca-1 + phenotype cells and CD34-Lin-c-kit + Sca-1 + phenotype cells in murine bone marrow cells increased (P <0.05). The single cell culture for 10 days showed that there was no significant difference in the number of clones formed per plate, the average number of cells per well and DAPT on the differentiation of single hematopoietic stem cells (P> 0.05). After 3 days of DAPT treatment, the ability of HSC in mouse bone marrow cells decreased. Conclusion: DAPT can accelerate the depletion of CD34-Lin-c-kit + Sca-1 + phenotype cells in mouse bone marrow cells and promote the expression of Lin-c-kit + Sca-1 + and CD34-Lin-c-kit + Sca-1 + phenotype cells, and decreased the ability of HSCs to proliferate in mouse bone marrow cells, but had no significant effect on the proliferation and differentiation of single CD34-Lin-c-kit + Sca-1 + phenotype cells.