CAJ | 학술논문

目的探讨细胞周期抑制剂PD0332991 (C24H29N7O2)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Hoechst33342染色和FITC-Annexin V/7-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G1期细胞明显增多,由76.3％增加至89.5％ (P＜0.05),S、G2/M期明显减少,由20.5％降低至7.3％ (P＜0.05)；HPC的G1期细胞也明显增多,由74％增加至87.4％ (P＜0.05),S、G2/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CDK1、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),凋亡相关基因及白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.
목적 탐토세포주기억제제PD0332991 (C24H29N7O2)대조혈간/조세포(HSPC)생물학행위적영향.방법 이용다항체표기,재분별용Hoechst33342염색화FITC-Annexin V/7-AAD표기류식세포술검측PD0332991작용전후소서골수조혈간세포(HSC,Lin-C-kit+Sca-1+표형세포)급조혈조세포(HPC,Lin-C-kit+세포)적주기분포급세포조망솔；이용실시형광정량PCR법분석PD0332991작용후HSC세포주기상관기인、조망상관기인급자아경신상관기인mRNA표체수평；이용겁한희석배양법검측PD0332991작용전후HSC자아확증수평적변화；용반고체배양법관찰PD0332991대소서정상HPC집락형성능력적영향.결과 여미처리조상비,PD0332991처리후소서골수HSC적G1기세포명현증다,유76.3％증가지89.5％ (P＜0.05),S、G2/M기명현감소,유20.5％강저지7.3％ (P＜0.05)；HPC적G1기세포야명현증다,유74％증가지87.4％ (P＜0.05),S、G2/M기세포명현감소,유25.4％강저지11.6％(P＜0.05).PD033299처리적소서골수HSPC조기조망솔균위7％좌우,여대조조상비차이무통계학의의(P＞0.05).소서골수HSC경PD0332991처리후,세포주기상관기인CDK1、CyclinA2、CyclinF、p18、p19、p27표체수평여대조조상비분별강저료58.8％、66.4％、56.3％、62.2％、32.3％화36.5％,CDK7표체상조27.3％ (P＜0.05),조망상관기인급백아확증상관기인표체균무명현변화.여대조조상비,PD0332991처리과적소서골수HSC여자아확증상관적아란석양구역형성세포출현빈솔명현하강、HPC적조혈집락형성능력하강.결론 PD0332991가명현조체정상소서HSPC적세포주기진정,억제기자아확증,단대기조망몰유명현영향.
Objective To investigate the effect of PD0332991 C24H29N7O2) on cell cycle,apoptosis,differentiation and self-renewal of hematopoietic stem cells (HSC) in mice.Method The selfrenewal 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).HPCsin G1 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.Conclusion 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 regulation of HSC/HPC.