Nucleostemin表达下调对p53缺失型HL-60细胞自噬活性的影响  

Effect of Down-Regulatory Nucleostemin Expression on Autophagy Activities in p53 Null HL-60 Leukemia Cells

作　　者：魏园玉 李曌博 张帆[1,3] 王宁宁[1,3] 刘帅[1,3] 岳保红[1,3] 

机构地区：[1]郑州大学第一附属医院检验科郑州大学医学检验系河南省高等学校临床医学重点学科开放实验室,河南郑州450052 [2]郑州大学第三附属医院检验科,河南郑州450052 [3]河南省检验医学重点实验室,河南郑州450052

出　　处：《中国实验血液学杂志》2017年第3期699-705,共7页Journal of Experimental Hematology

基　　金：国家自然科学基金资助项目(81271911);河南省医学科技攻关重点资助项(201002006)

摘　　要：目的:结合前期基因芯片和生物信息学分析结果,探讨下调Nucleostemin(NS)表达对p53缺失型白血病HL-60细胞自噬活性的影响,为更深入研究NS非p53依赖性信号通路的关键节点提供依据。方法:在NS-RNAiGV248重组慢病毒载体下调NS表达的基础上,采用吖啶橙染色、Western blot和透射电镜技术,检测HL-60细胞自噬活性的变化。结果:重组慢病毒载体成功抑制了HL-60细胞中NS蛋白的表达;与空白对照组(3.1±0.28)%和阴性对照组(6.2±0.64)%相比,实验组HL-60细胞内酸性小体数量(22.4±0.76)%明显增高(P<0.05);与阴性对照组(1.010±0.039)和空白对照组(0.608±0.008)相比,实验组HL-60细胞LC3Ⅱ/LC3Ⅰ的比值(1.537±0.072)明显增高(P<0.05);与阴性对照组(4.2±1.2)和空白对照组(2.3±0.5)相比,实验组中HL-60细胞内自噬性结构的数量(8.7±3.1)明显增高(P<0.05)。结论:下调NS蛋白的表达可以增强p53缺失型HL-60细胞的自噬活性。Objective： Based on previous microarry and bioinformatic analysis results, to investigate the effect of nucleostemin（NS） expression down-regulation on autophagy activity in p53 null HL-60 leukemia cells, so as to provide evidence for studying mechanisms of p53-independent signal pathway of NS in details. Methods ： The autophagy activity of HL-60 cells after down-regulation of NS expression was detected with acidine orange staining, Western blot and trans- mission electron mcrioscope technique. Results： The expression level of NS in test groups was lower than that in blank control and negative control groups after HL-60 cells were readily transinfected by lentivirus. The result of acidine orange staining showed that the number of acid vesicular organelle in test groups （ 22.4± 0.76 ） % was higher than that in blank control groups （ 3.1 ± 0.28 ） % and negative control groups （ 6.2± 0.64 ） % （ P 〈 0.05 ）. Western blot showed that the ra- tio of LC3 II/LC3 I in test groups （ 1. 537 ± 0. 072 ） was higher than that in blank control and negative control groups （1. 010 ±0. 039） and （0.608 ±0. 008）. The result of transmission electron mcrioscopy also showed that the number of autophagosomes in test group （ 8.7 ± 3.1 ） was higher than that in the blank control and negative control groups （4.2 ± 1.2） and （2.3 ±0.5 ）. Conclusion ： Autophagy activty can be enhanced after the level of NS was down regulated. The change indicates the signaling transductions screened by bioinformatic analysis may be one of p53-independent pathway of NS, which lays a foundation for contineously studying key points of p53-independent signal pathway of NS.

关 键 词：NUCLEOSTEMIN HL-60细胞 非P53依赖性通路 自噬活性 

分 类 号：R733.71[医药卫生—肿瘤]