重组慢病毒表达PML核定位信号缺失体抑制急性早幼粒白血病细胞系c-Myc蛋白表达

The expression of c-Myc protein in acute promyelocytic leukemia cell lines is inhibited by recombinant lentivirus expressing PML( ΔNLS)

目的构建携带PML(ΔNLS)基因的重组慢病毒,研究其对NB4细胞中c-Myc蛋白表达的影响。方法以质粒p CMV-HA-PML(ΔNLS)为模板,PCR扩增PML(ΔNLS)基因,克隆入慢病毒载体LV5-EF1a-GFP/puro,与p Helper 1.0和p Helper 2.0共转染293T细胞,包装病毒。收取病毒上清液,纯化后感染NB4细胞,荧光倒置显微镜观察感染效率,RT-PCR法检测PML(ΔNLS)mRNA的转录水平,Western blot检测PML(ΔNLS)、β-catenin、γ-catenin和c-Myc蛋白表达的变化。CCK-8实验观察细胞增殖。结果成功构建PML(ΔNLS)过表达慢病毒,病毒感染NB4细胞,感染效率达82.74%,LV5-PML(ΔNLS)携带的PML(ΔNLS)能够在NB4细胞中稳定表达;感染LV5-PML(ΔNLS)的NB4细胞中β-catnin、γ-catenin和c-Myc蛋白表达下降(P<0.05);NB4细胞增殖活力明显高于未感染组和空病毒载体组(P<0.05)。结论成功构建了重组慢病毒PML(ΔNLS),PML(ΔNLS)过表达可促进NB4细胞体外增殖,并抑制c-Myc蛋白表达。

Objective To construct recombinant lentivirus carrying PML（ ΔNLS） gene and to observe its effect on the expression of c-Myc protein in leukemia NB4 cell lines. Methods PML（ ΔNLS） gene was amplified by PCR using plasmid p CMV-HA-PML（ ΔNLS） as a template and cloned into the lentivirus vector LV5-EF1a-GFP / puro.The recombinant lentiviral vector,pHelper 1. 0 and pHelper 2. 0 were co-transfected into 293 T cells,packaging virus. Then NB4 cells were infected with the supernatant containing lentiviral particles,and its infection efficiency was determined by fluorescent microscope,PML（ ΔNLS） mRNA transcription and protein expression in NB4 cells were determined by RT-PCR and Western blot respectively,the expression of β-catenin,γ-catenin and c-Myc proteins in NB4 cells were analyzed by Western blot. Cell proliferation was detected by CCK-8. Results Recombinantlentivirus LV5-PML（ ΔNLS） was successfully constructed. The infection efficiency in NB4 cells reached82. 74 %,the PML（ ΔNLS） gene in LV5-PML（ ΔNLS） was expressed stably in NB4 cells; the expression ofβ-catenin,γ-catenin and c-Myc proteins were decreased in NB4 cells infected with LV5-PML（ ΔNLS）. The activity of cell proliferation in NB4 cells significantly enhanced as compared to the untransfeced and empty viral groups. Conclusions Recombinant lentivirus LV5-PML（ ΔNLS） was successfully constructed and infected NB4 cells. In addition,it may inhibit the expression of c-Myc protein.