DAPT对小鼠造血干细胞生物活性的影响  

Effects of DAPT on Biological Activity of Hematopoietic Stem Cells in Mice

作　　者：张娜 张丽艳 张燕 纪庆 王金宏 祁瑞哲 许静 袁卫平 程涛 高瀛岱 

机构地区：[1]中国医学科学院、北京协和医学院血液学研究所、血液病医院实验血液学国家重点实验室,天津300020

出　　处：《中国实验血液学杂志》2012年第3期679-685,共7页Journal of Experimental Hematology

基　　金：国家自然科学基金(编号81170465,90913018,81090414,30825017);实验血液学国家重点实验室开放课题基金(编号ZK11-07,ZK08-04)

摘　　要：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（P0.05） 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 G0 phase decreased and at G1 phase increased after treatment with DAPT 1 μmol/L for 5 d（P0.05） 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.

关 键 词：NOTCH信号通路 造血干细胞 DAPT 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]