过表达prohibitin促进NB4-R1急性早幼粒细胞白血病维甲酸耐药细胞凋亡  

Over-expression of prohibitin gene promotes apoptosis in retinoic acid-resistant acute promyelocytic leukemia cell line NB4-R1

作　　者：刘雁峰[1] 贺鹏程[1] 刘锋[1] 程小岩[1] 张梅[1] 

机构地区：[1]西安交通大学医学院第一附属医院血液内科,陕西西安710061

出　　处：《细胞与分子免疫学杂志》2014年第9期933-936,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：陕西省科技统筹创新工程计划项目(2012KTCL03-12)

摘　　要：目的构建人prohibitin基因真核过表达载体,探讨上调prohibitin对NB4-R1急性早幼粒细胞白血病维甲酸耐药细胞凋亡的影响。方法 PCR钓取目的基因prohibitin,与pEGFP-N1-3FLAG真核载体定向连接,PCR筛选阳性克隆,测序鉴定。将阳性重组载体pEGFP-N1-3FLAG-prohibitin转染NB4-R1细胞,以实时定量PCR(qRT-PCR)和Western blot法检测过表达效果,annexin V-FITC/PI染色结合流式细胞术检测prohibitin过表达对NB4-R1细胞凋亡的影响。结果 PCR筛选及测序比对,证明成功构建了prohibitin基因过表达载体;对NB4-R1细胞转染率可达70%以上;qRT-PCR结果显示转染pEGFP-N1-3FLAG-prohibitin过表达载体组(OE)prohibitin mRNA表达水平较空白对照组(CON)和阴性对照组(NC)分别上调(1.64±0.37)倍和(1.58±0.43)倍(P<0.05),Western blot结果显示OE组prohibitin蛋白表达水平较CON组和NC组分别上调(1.91±0.33)倍和(1.99±0.37)倍(P<0.05);流式细胞术结果表明CON组、NC组和OE组NB4-R1细胞凋亡率分别为(5.88±0.43)%、(6.63±0.46)%和(28.22±1.33)%,OE组较CON组和NC组细胞凋亡率分别增加了(3.80±0.24)倍和(3.39±0.30)倍(P<0.05)。结论过表达prohibitin可诱导NB4-R1细胞凋亡。Objective To construct a eukaryotic expression vector carrying human prohibitin gene and study the effect of prohibitin over-expression on the apoptosis of retinoic acid-resistant acute promyelocytic leukemia NB4-R1 cells. Methods The prohibitin gene was amplified by PCR,and then cloned into pEGFP-N1-3FLAG eukaryotic expression vector. Positive clones were selected by PCR screening and identified by DNA sequencing. The positive recombinant vector pEGFP-N1-3FLAG-prohibitin was transferred into NB4-R1 cells. The over-expression of prohibitin was verified by real-time PCR and Western blotting,respectively. The apoptosis rate of the NB4-R1 cells transfected with pEGFP-N1-3FLAG-prohibitin was measured by flow cytometry combined with annexin V-FITC /PI staining. Results PCR screening and DNA sequencing demonstrated that the recombinant plasmid of pEGFP-N1-3FLAG-prohibitin was constructed successfully; and the transfection efficiency of NB4-R1 cells reached more than 70%. Moreover,qRT-PCR showed that the expression of prohibitin mRNA of transfection group（ OE） increased by（ 1. 64 ± 0. 37） times and（ 1. 58 ± 0. 43） times（ P〈0. 05） compared with blank control group（ CON） and negative control group（ NC）,respectively. Western blotting showed that the expression of prohibitin protein of OE group increased by（ 1. 91 ± 0. 33） times and（ 1. 99 ± 0. 37） times（ P〈 0. 05） compared with CON and NC,respectively; Flow cytometry indicated that the NB4-R1 cell apoptosis rates in CON,NC and OE groups were respectively（ 5.88 ±0.43） %,（ 6.63 ±0.46） % and（ 28.22 ±1.33） %,and the apoptosis rate of OE group was elevated by（ 3.80 ±0.24）times and 3. 39 ± 0. 30 times compared with CON and NC（ P〈 0. 05）,respectively. Conclusion The up-regulated expression of prohibitin could promote NB4-R1 cell apoptosis.

关 键 词：PROHIBITIN 过表达 NB4-R1细胞 凋亡 急性早幼粒细胞白血病 

分 类 号：R733.71[医药卫生—肿瘤] R392-33[医药卫生—临床医学]