Objective: To investigate the effects of IRF1 on the homeostasis and differentiation of K562 cells.
Methods: Three different vectors were constructed to screen the best strategy for IRF1 overexpression. The effect of IRF1 on cell proliferation and apoptosis was explored by cell count and apoptotic surface marker detection. Likely, the effect of IRF1 on cell differentiation was analyzed by differentiational surface marker assay. Finally, the regulation mechanism at mRNA level was analyzed by RT-qPCR.
Results: The single open reading frame constructed by P2A-T2A element showed the highest expression intensity, and it was the best approach to realize IRF1 enhancement. Cell counts showed that IRF1 had no significant effect on the proliferation of K562. Annexin V and 7-AAD labeling exhibited strong anti-apoptotic function of IRF1 against AraC induction. Flow cytometry revealed that IRF1 overexpression could also further increase the proportion of CD71+CD235a+ cells. RT-qPCR confirmed its upregulation effect on CD235a and TAL1.
Conclusion: IRF1 enhancement alters the homeostasis characteristics of K562 cells, increases the anti-apoptotic ability and raises the potential to downstream differentiation, suggesting that IRF1 may play an important regulatory role in the hematopoietic development, including erythropoiesis.
题目: 过表达干扰素调节因子IRF1重塑K562细胞的稳态特征.
目的: 考察干扰素调节因子IRF1对K562细胞稳态及分化的影响.
方法: 构建3种不同的过表达载体以筛选实现IRF1增强表达的最佳方式；通过细胞计数考察IRF1对细胞增殖的影响；通过凋亡标记分析IRF1对药物诱导凋亡的影响；通过分化标记分析IRF1对红系分化的影响；通过RT-qPCR初步探索IRF1的调控机制.
结果: 利用P2A-T2A方式构建的单一开放阅读框具有最明显的表达效果，是实现IRF1增强表达的最佳方式；细胞计数结果发现，IRF1对K562的增殖无明显影响；Annexin V和7-AAD联合标记发现，IRF1过表达显著抑制细胞的凋亡进程；流式结果显示，IRF1同样可进一步提升CD71+CD235a+双阳细胞比例，RT-qPCR检测证实了IRF1过表达对CD235a的调控作用.
结论: IRF1过表达改变了K562细胞的稳态特征，增强细胞的抗凋亡能力并促进了向下游分化的潜力，提示IRF1在红系系统发育中可能发挥重要的调控功能.