基础医学与临床 ›› 2014, Vol. 34 ›› Issue (10): 1327-1332.

• 研究论文 • 上一篇    下一篇

腺病毒介导的PML(NLS-)过表达对白血病NB4细胞增殖和凋亡的影响

阳小群,王慧,蒋开玲,朱新瑜,马鹏鹏,刘北忠   

  1. 重庆医科大学
  • 收稿日期:2014-03-13 修回日期:2014-05-26 出版日期:2014-10-05 发布日期:2014-09-25
  • 通讯作者: 刘北忠 E-mail:lbz2753@qq.com
  • 基金资助:
    APL诊疗新靶点的筛选与研究

Effect of adenovirus-mediated PML (NLS-) over-expression on the proliferation and apoptosis of NB4 leukemic cells

  • Received:2014-03-13 Revised:2014-05-26 Online:2014-10-05 Published:2014-09-25

摘要: 目的 利用腺病毒介导PML(NLS-)的过表达,探讨PML(NLS-)对NB4白血病细胞增殖凋亡的影响。方法 重组腺病毒质粒Ad-PML(NLS-)经Pac I酶切线性化后转染AD293细胞,获得重组腺病毒Ad-PML(NLS-),经4轮扩增后,测定重组腺病毒滴度;重组腺病毒感染NB4细胞,荧光显微成像和流式细胞术检测感染效率,RT-PCR法和Western blot法检测NB4细胞中PML(NLS-)的转录及表达水平,MTT实验观察细胞增殖,流式细胞术分析细胞周期及凋亡。结果 重组腺病毒质粒Ad-PML(NLS-)经酶切和测序鉴定正确,经4轮扩增病毒滴度为 ;重组腺病毒Ad-PML(NLS-)感染NB4细胞效率达75%,PML(NLS-)基因可在NB4细胞中高效表达; NB4细胞增殖活力明显高于未感染组和空病毒载体感染组(p<0.05),S期细胞比例明显增高(p<0.05),G2期细胞比例明显减少(p<0.05);实验组细胞凋亡率明显低于空病毒感染组(p<0.05)。结论 PML(NLS-)过表达能够促进白血病NB4细胞的体外增殖能力,抑制其凋亡。

关键词: PML(NLS-), 急性早幼粒细胞白血病, 增殖, 凋亡

Abstract: Objective To investigate the effect of PML (NLS-) on proliferation and apoptosis of leukemic NB4 cells by the over-expression of PML(NLS-) induced by adenoviruses. Methods Recombinant adenovirus plasmid Ad-PML (NLS-) was digested and linearized with Pac I enzyme then transfected into AD293 cells. After amplified the Recombinant adenovirus Ad-PML(NLS-) for four times, to test recombinant adenovirus titer. After Recombinant adenovirus transfected into NB4 cells, infection efficiency was detected by fluorescence microscopy imaging and flow cytometry, the transcription and expression level of PML(NLS-) in NB4 cells were detected by RT-PCR and Western blot. Cell proliferation was detected by MTT. Cell cycle and apoptosis was detected by flow cytometry. Results Recombinant adenovirus plasmid Ad-PML (NLS-) was exactly digested and sequenced. The titer of amplified virus was . The efficiency of transfection was 75%, and PML(NLS-) had a high level expression in NB4 cells. The activity of cell proliferation in NB4 cells was significant enhanced compared with the untransfected and empty viral vector (p<0.05). The proportion of cells in S phase was obviously increased while which was obviously decreased in G1 phase. The rate of apoptosis in the experimental group was obviously lower than the empty viral vector. Conclusions The over-expression of PML(NLS-) could promote the proliferation of NB4 cells in vitro and inhibit the apoptosis.

Key words: PML(NLS-), acute promyelocytic leukemia, proliferation, apoptosis