细胞周期抑制剂PD0332991对小鼠造血干／祖细胞生物学活性的影响

Effect of PD0332991 on biological activity of hematopoietic stem cells in mice

目的探讨细胞周期抑制剂PD0332991（C24H29N702）对造血干／祖细胞（HSPC）生物学行为的影响。方法利用多抗体标记，再分别用Hoechst 33342染色和FITC-AnnexinW7-AAD标记流式细胞术检测PD0332991作用前后小鼠骨髓造血干细胞（HSC，Lin-C-kit＋Sca-1＋表型细胞）及造血祖细胞（HPc，Lin-C-kit’细胞）的周期分布及细胞凋亡率；利用实时荧光定量PCR法分析PD0332991作用后HSC细胞周期相关基因、凋亡相关基因及自我更新相关基因mRNA表达水平；利用极限稀释培养法检测PD0332991作用前后HSC自我扩增水平的变化；用半固体培养法观察PD0332991对小鼠正常HPC集落形成能力的影响。结果与未处理组相比，PD0332991处理后小鼠骨髓HSC的G。期细胞明显增多，由76．3％增加至89．5％（P〈0．05），S、GYM期明显减少，由20．5％降低至7．3％（P〈0．05）；HPC的G。期细胞也明显增多，由74％增加至87．4％（P〈0．05），S、G：／M期细胞明显减少，由25．4％降低至11．6％（P〈0．05）。PD033299处理的小鼠骨髓HSPC早期凋亡率均为7％左右，与对照组相比差异无统计学意义（P〉0．05）。小鼠骨髓HSC经PD0332991处理后，细胞周期相关基因CDKl、CyclinA2、CyclinF、p18、p19、p27表达水平与对照组相比分别降低了58．8％、66．4％、56．3％、62．2％、32．3％和36．5％，CDK7表达上调27．3％（P〈0．05），凋亡相关基因及自我扩增相关基因表达均无明显变化。与对照组相比，PD0332991处理过的小鼠骨髓HSC与自我扩增相关的鹅卵石样区域形成细胞出现频率明显下降、HPC的造血集落形成能力下降。结论PD0332991可明显阻滞正常小鼠HSPC的细胞周期进程，抑制其自我扩增，但对其凋亡没有明显影响。

Objective To investigate the effect of PD0332991 （C24H29N702） on cell cycle, apoptosis, differentiation and self-renewal of hematopoietic stem cells （HSC） in mice. Method The self- renewal ability of HSCs was measured by cobblestone forming cell assay （CAFC）. The colony-forming cell （CFC） assay was used to quantify the changes of numbers and functions of HPC after the treatment of the compound. The expressions of self-renewal regulation genes, cell cycle-related genes, apoptosis-related genes were measured by real-time PCR. The cell cycle status and apoptosis of HSC and HPC were analyzed by flow cytometry. Result There were obvious changes in cell cycle regulation between control and PD0332991 groups. HSCs in G1 phase increased significantly from 76.3% to 89.5% after treatment of PD0332991 （P〈0.05） while cells in S, G2 and M phase reduced from 20.5% to 7.3% （P〈0.05）. HPCs in G, phase also increased from 74% to 87.4% after treatment of PD0332991 （P〈0.05） while cells in S, G2 and M phase reduced from 25.54% to 11.6% （P〈0.05）. The apoptotic fractions between control and PD0332991 groups had no statistical difference （P〉0.05）. After cultured with PD0332991, the expression levels of cell cycle genes CDK1, CyclinA2, CyclinF, p18, p19 and p27 decreased by 58.77%, 66.35%, 56.33%, 62.18%, 32.28% and 36.53% respectively, while expression of CDK7 increased by 27.27% （P〈0.05）. No visible expression difference was observed in apoptosis and self-renew related genes. After treatment of PD0332991, the self-renewal ability of HSC decreased significantly. There were almost no CFCs in PD0332991 group in CAFC assay. Similarly, the frequency of CFCs was dramatically lower in PD0332991 group. Conelusion These results suggested that PD0332991 affected HSC/HPC from mice mainly through inhibiting the cell cycle rather than apoptosis. It also suggested that CDK4/6 might play a key role in the reeulation of HSC/HPC.