RESUMO
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 CD71CD235a 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.
RESUMO
OBJECTIVE@#To compare the efficacy of directional erythroid differentiation in different serum free culture systems and to screen the optimal culture systems for inducing the differentiation of umbilical cord blood hematopoietic stem and progenior cells (HSPC) to erythroid cells.@*METHODS@#The CD34 cells from umbilical blood munonuclear cells were sorted by using the magnetic beads, and were inoculated into 3 different of culture systems (system 1, 2 and 3 respectively), to induce erythrold differentiation by 3 stage culture. The living cells were counted in different differentiation stages and were observed by Wright-Giemsa staining; the expression of CD71 and CD235a on cell surface was detected by flow cytometry, the erythroid differentiation pteency was detected via colony-forming test.@*RESULTS@#The ability of system 2 to promote the HSPC proliferation was the strongest, the efficacy of system 3 to promote the erythroid differentiation of HSPC was the most optimal; the proliferation ability of cells cultured in system 2 for 2-15 days all was higher than that of cells cutured in system 1 and 3 (P＜0.05). The flow cytometry detection showed that the expression of CD71 and CD235a on surface of cells cultured in system 3 was the highest, the CD235a percentage on day 15 of differentiation in system 3 was (92.33±3.89)%, that in system 2 was (84.67±3.12)%, while that in system 1 was (72.17±6.83)% (P＜0.05). Cell morplologic detection showed that throid differentiation was accelerated on day 12, the percentage of orthochromatic erythrocytes in system 3 was (67.67±2.08)% which was 10.69 and 25.34 times higher than that in system 2 and 1 respectively (P＜0.05). The colony-forming test showed the ratio of BFU-E in system 3 increased gradually on day 3-9 (r=0.99, P＜0.05), which was significanlly higher than that in system 2 and 1 on day 9 (90.35±5.52% vs 77.06±2.26% and 74.50±3.95%).@*CONCLUSION@#Culture system 3 is the most effective serum-free erythroid differentiation system, and the culture system 2 is the most powerful HSPC proliferation system. This study results provide a technical basis for further efficiently increasing and inducing the erythroid proliferation and differentiation of HSPC, and also provide culture system in vitro for the clinical application and basic research.