P21基因参与早期诱导RUNX1b过表达抑制人类造血发生

P21 gene is involved in early inducible overexpression of RUNX1b and blocks the human hematopoiesis

目的探究细胞周期蛋白依赖性激酶抑制剂1[CDKN1A(P21)]对造血发生过程的影响。方法对RUNX1b诱导过表达hES细胞系(RUNX1b/hESC)与小鼠主动脉-性腺-中肾区(AGM)基质细胞AGM-S3共培养4 d细胞,使用多西环素(DOX)(1μg/mL)与RepSox(0.33μmol/L)或不同浓度DOX(1、0.5、0.2、0.1μg/mL)诱导RUNX1b过表达时,以qRT-PCR技术检测P21与RUNX1b相对表达情况。利用基于转座子载体PiggyBac的PB-tet-on-OE真核诱导过表达系统,建立P21诱导过表达hES细胞系(CDKN1A/hESC)。分别在CDKN1A/hESC与AGM-S3共培养d0、d2、d4、d6添加DOX诱导P21过表达,培养8 d或14 d,通过流式细胞术(FACS)检测血细胞表面分子的变化情况,以判断诱导P21过表达对造血发生产生的影响。结果 qRT-PCR显示:P21表达量随着RUNX1b过表达上调,同时加入RepSox时表达基本处在正常水平,随着DOX浓度降低,RUNX1b与P21表达水平下降。在造血分化的早期,特别是共培养d0开始诱导P21过表达的共培养体系所产生的CD34^-CD43^+、CD34^-CD45^+与CD34^+CD45^+细胞群相较于对照分别降低了85%,94%和78%,当共培养d6后诱导P21过表达上述现象消失。结论 P21可能参与了由RUNX1b诱导过表达所引起的人类早期造血发生的抑制效应;早期诱导P21过表达明显抑制造血细胞产生。

Objective To investigate the effect of cyclin-dependent kinase inhibitor 1[CDKN1A(P21)]on hematopoiesis.Methods The co-cultured d4 cells of inducible RUNX1b hESC line(RUNX1b/hESC)with mouse aorta/gonad/mesonephros(AGM-S3),using doxycycline(DOX)(1μg/mL)and RepSox(0.33μmol/L)or different concentrations of DOX(1,0.5,0.2,and 0.1μg/mL)to induce RUNX1b overexpression,qRT-PCR was used to detect the relative expression of P21 and RUNX1b.Eukaryotic inducible system based on piggyBac,PB-tet-on-OE,was used to establish inducible P21 hESC line(CDKN1A/hESC).DOX was added in CDKN1A/hESC and co-cultured with AGM-S3 from 0,2,4 d,and d6 respectively,to induce P21 overexpression.At co-cultured d8 and d14,fluorescence-activated cell sorting(FACS)was used to detect changes of surface molecules of hematopoietic cells,and investigate the effect of P21 overexpression on hematopoiesis.Results qRT-PCR showed that the expression of P21 was up-regulated together with RUNX1b overexpression,and the expression of P21 was restored to the normal level when RepSox was added.As the concentration of DOX decreased,the expression of RUNX1b and P21 also decreased.Overexpression of P21 from early stage of hematopoiesis,especially CD34^-CD43^+,CD34^-CD45^+,CD34^+CD45^+populations from 0 d,decreased by 85%,94%,and 78%,respectively,when compared with the control group,but the above overexpression disappeared when induced after 6 days.Conclusion P21 is probably involved in inhibitory effect of human hematopoiesis caused by overexpression of RUNX1b at early stage;induction of P21 overexpression from early stage significantly blocks the product of hematopoietic cells.