NLS-RARα通过激活AKT调节白血病NB4细胞的增殖  

NLS-RARα regulates proliferation of leukemia cell line NB4 through the activation of AKT

作　　者：宋浩[1,2] 李浏[2] 钟梁[2] 蒋开玲[2] 阳小群[2] 杨蓉[2] 刘北忠[1,2] 

机构地区：[1]重庆医科大学附属永川医院中心实验室,重庆402160 [2]重庆医科大学临床检验诊断学教育部重点实验室,重庆400016

出　　处：《基础医学与临床》2016年第1期41-46,共6页Basic and Clinical Medicine

基　　金：国家自然科学基金(81171658);重庆市自然科学基金计划重点项目(2011BA5037)

摘　　要：目的构建携带NLS-RARα的重组慢病毒,感染白血病NB4细胞并观察其对NB4细胞增殖和AKT蛋白的影响。方法以质粒p CMV-HA-NLS-RARα为模板,PCR扩增NLS-RARα基因,将其亚克隆到慢病毒表达载体质粒LV5中,经DNA测序鉴定重组载体,用4质粒系统共转染293T细胞,收集病毒上清,浓缩鉴定,测定重组慢病毒的滴度;重组慢病毒感染NB4细胞,经过嘌呤霉素筛选获得稳定转染NLS-RARα的NB4细胞株;CCK-8法检测NLSRARα对NB4细胞增殖的影响;流式细胞术检测细胞周期和感染效率;Western blot分别检测NLS-RARα、t-AKT和p-AKT的表达。结果 NLS-RARα克隆入LV5的多克隆位点,慢病毒浓缩后滴度为2×108Tu/m L;感染效率为86.54%;NLS-RARα能够明显促进细胞增殖,S期细胞增多,G1和G2期细胞减少;NLS-RARα基因在NB4细胞中稳定的表达,同时p-AKT明显增高,用PI3K抑制剂LY294002处理后p-AKT明显降低。结论 NLS-RARα通过激活AKT促进白血病NB4细胞的增殖。Objective To construct NLS-RARα recombinant lentivirus and detect its effect on proliferation and expression of AKT protein of leukemia cell line NB4. Methods NLS-RARα gene was amplified by PCR with plasmid p CMV-HA-NLS-RARα as a template. The gene of NLS-RARα was subcloned into the shuttle plasmid LV5-EF1 aGFP / PURO by DNA recombinant technique. The four plasmid systems were transfected into 293 T cells by liposome. The recombinant lentivirus vector of NLS-RARα was used to infect Leukemia cell line NB4. Cell viability was evaluated 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 × 10^8 Tu / mL. The infection efficiency of LV5-NLS-RARα in NB4 cells reached 86. 54%. The proliferation of NB4 cellswhich over-expressed NLS-RARα was enhanced. The proportion of cells in S phase was increased but was decreased in G1 and G2phases of the cell cycle. The protein level of NLS-RARα and p-AKT was significantly increased. The level of p-AKT decreased after pretreatment with PI3 K inhibitor LY294002. Conclusions NLSRARα prometos proliferation of leukemia cell line NB4 through the activation of AKT.

关 键 词：NLS-RARα 急性早幼粒细胞白血病 增殖 AKT 

分 类 号：R733.71[医药卫生—肿瘤]