关键词: NLS-RARα, 急性早幼粒细胞白血病, 增殖, AKT

Abstract: Objective To construct NLS-RARα recombinant lentivirus and detect its effect on proliferation and AKT protein of leukemia cell line NB4. Methods NLS-RARα gene was amplified by PCR using plasmid pCMV-HA-NLS-RARα as a template. The resulting gene of NLS-RARα was subcloned into the shuttle plasmid LV5-EF1a-GFP/PURO by using DNA recombinant technique. The four plasmids system was transfected into 293T cells by liposome. The virus supernatant was harvested and concentrated, then determined the titer. The recombinant lentivirus vector of NLS-RARα was used to infect Leukemia cell line NB4, the stably transfected cell lines were selected by puromycin. Cell viability was detected by CCK-8 assay. Cell cycle and infection efficiency were measured by flow cytometry. The expression of NLS-RARα, t-AKT and p-AKT were detected by Western blot. Results Recombinant lentivirus plasmid LV5-NLS-RARα was constructed correctly as proved by DNA sequencing. The titer of the lentivirus reached 2×108 Tu/mL. The infection efficiency of LV5-NLS-RARα in NB4 cells reached 86.54%. The proliferation of NB4 cells which over-expressed NLS-RARα was enhanced. The proportion of cells in S phase was increased which was decreased in G1 and G2 phases of the cell cycle. The protein level of NLS-RARα and p-AKT were significantly increased. The level of p-AKT was decreased after pretreatment with PI3K inhibitor LY294002. Conclusion NLS-RARα prometos proliferation of leukemia cell line NB4 through the activation of AKT.

Key words: NLS-RARα, acute promyelocytic leukemia, proliferation, AKT