Puma表达下调对人脐血CD34＋造血细胞抗放射作用的初步研究

Knockdown of Puma Protects Cord Blood CD34^+ Cells againstγ-Irradiation

Puma属于BCL-2家族中BH3-only亚家族,动物和细胞研究发现其通过p53依赖和非依赖途径诱导细胞凋亡,并具有抗放射保护不增加肿瘤发生率的作用。本研究检测Puma基因敲降后对人脐血CD34+造血干祖细胞生物学功能的影响。应用RT-PCR、Western Blot检测Puma基因在辐射刺激条件下的表达,采用慢病毒感染人脐血CD34+细胞分选GFP+细胞,Annexin V-PE/7-AAD双染流式细胞术检测细胞凋亡,Ki67染色检测细胞周期,CCK-8、Brdu标记法检测细胞增殖能力,并进行集落形成实验(colony formig cell assay,CFC)。结果表明:Puma基因表达量随着射线强度的增加而增高,Puma基因表达下调抑制造血祖细胞体外集落形成能力和体外增殖能力。在辐射刺激条件下,抑制Puma基因表达可以减少人脐血CD34+细胞凋亡,维持细胞周期于G0期。结论:人脐血CD34+造血干细胞中Puma基因敲降具有一定的抗辐射作用,维持细胞于静息状态。

Puma （P53 upregulated modulator of apoptosis） is a BCL-2 homology 3 （BH3）-only BCL-1 family member and a critical mediator of P53-dependent and - independent apoptosis. Puma plays an essential role in the apop- tosis of hematopoietic stem cells exposed to irradiation without an increased risk of malignancies. This study was pur- posed to develop an effective lentiviral vector to target Puma in human hematopoietic cells and to investigate the effect of Puma gene knockdown on the biological function of human cord blood CD34 ＋ cells. SF-LV-shPuma-EGFP and control vectors were constructed, and packaged with the pSPAX2/pMD2. G packaging plasmids via 293T cells to produce pseu- do-type lentiviruses. SF-LV-shPuma-EGFP or control lentiviruses were harvested within 72 hours after transfection and then were used to transduce human cord blood CD34＋ cells. GFP ＋ transduced cells were sorted by flow cytometry （FCM） for subsequent studies. Semi-quantitative real time RT PCR, Western blot, FCM with Annexin V-PE/7-AAD double staining, Ki67 staining, colony forming cell assay （CFC）, CCK-8 assay and BrdU incorporation were performed to determine the expression of Puma and its effect on the cord blood CD34 ＋ cells. The results showed that Puma was significantly knocked down in cord blood CD34 ＋ cells and the low expression of Puma conferred a radio-protective effect on the cord blood CD34 ＋ cells. This effect was achieved through reduced apoptosis and sustained quiescence after irradi- ation due to Puma knockdown. It is concluded that knockdown of puma gene in CD34 ＋ hematopoietic stem cells of hu- man cord blood possesses the radioprotective effect, maitains the cells in silence targeting Puma in human hematopoietic cells may have a similar effect with that on mouse hematopoietic cells as previously shown, and our lentiviral targeting system for Puma provides a valuable tool for future translational studies with human cells.