DAPT对小鼠造血干细胞生物活性的影响

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。

Effects of DAPT on biological activity of hematopoietic stem cells in mice

This study was to investigate the effects of DAPT (N-[N-(3,5-difluorophenacetyl-L:-alanyl)]-S-phenylglycinet-butyl ester) on cell cycle, apoptosis, differentiation and expansion of hematopoietic stem cells (HSC) of mouse and to elucidate the possible mechanisms. The mRNA expressions of cell cycle-related genes p18, p21, p27, CDK1, CDK2, CDK4, CDK6, and apoptosis-related genes Bcl-2, Bcl-xl, mcl-1, Bax, Bim, p53, Puma were measured by real-time PCR. The cell cycle and apoptosis of Lin(-)c-kit(+)Sca-1(+) marked cells and CD34(-)Lin(-)c-kit(+)Sca-1(+) marked cells in bone marrow cells were detected by flow cytometry. The differentiation level of HSC was determined by single cell culture. The expansion of HSC were measured with long-term culture. The results indicated that the mRNA expression of the cell cycle related-genes CDK1, CDK2, CDK4, CDK6, p27 in Lin(-) c-kit(+)Sca-1(+) marked cells increased (P < 0.05), the expression of p18, p21 decreased (P < 0.05), the expression of the apoptosis related-genes Bcl-2, Bcl-xl, Bax, p53, Puma in Lin(-) c-kit(+)Sca-1(+) marked cells increased (P < 0.05), the expression of Bim decreased (P < 0.05), the expression of Mcl-1 had not changed (P > 0.05) after treatment with DAPT 1 μmol/L for 5 d. The changes of cell cycle of Lin(-)c-kit(+)Sca-1(+) marked cells in bone marrow had no statistical significance after treatment with DAPT 1 μmol/L for 5 d, CD34(-)Lin(-)c-kit(+)Sca-1(+) marked cells in bone marrow at G(0) phase decreased and at G(1) phase increased after treatment with DAPT 1 μmol/L for 5 d (P < 0.05); the apoptotic fractions of Lin(-) c-kit(+) Sca-1(+) marked cells and CD34(-)Lin(-)c-kit(+)Sca-1(+) marked cells in bone marrow increased after treatment with DAPT 1 μmol/L for 5 d (P < 0.05). The changes of colony number, average number of cells in wells and their differentiation had no statistical significance (P > 0.05) after treatment with DAPT 1 μmol/L for 10 d. Expansion of HSC in bone marrow of mouse decreased after treatment with DAPT 1 μmol/L for 3 d. It is concluded that DAPT not only enhances the exhaustion of CD34(-)Lin(-)c-kit(+)Sca-1(+) marked cells in bone marrow cells of mouse, but also enhances the apoptosis of Lin(-)c-kit(+)Sca-1(+) marked cells and CD34(-)Lin(-)c-kit(+)Sca-1(+) marked cells in bone marrow cells of mouse. DAPT also reduces the expansion of HSC. However, the changes of survival and differentiation of single CD34(-)Lin(-)c-kit(+)Sca-1(+) marked cells in mouse bone marrow cells have no statistical significance.