干扰核仁磷酸蛋白对人白血病OCI／AML3细胞凋亡的影响及其机制  

Effect of nucleophosmin(NPM) 1 knockdown on the apoptosis of OCI/AML3 leukemic cell with mutant NPM1 and its mechanisms

作　　者：王娟[1] 陈莎娜[1] 全静[1] 贺金刚[1] 张帅帅[1] 鲜敬荣[1] 张伶[1] 

机构地区：[1]重庆医科大学检验医学院血液教研室、临床检验诊断学教育部重点实验室、重庆市重点实验室,重庆400016

出　　处：《重庆医科大学学报》2014年第11期1578-1583,共6页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:81271913);重庆市留学人员科技活动择优资助项目(编号:市级渝留助2013009)

摘　　要：目的:探讨抑制核仁磷酸蛋白(nucleophosmin,NPM)1基因对人白血病细胞凋亡的影响及相关机制。方法:将空载慢病毒p GIPZ和NPM干扰慢病毒sh NPM分别感染携带NPM1突变的OCI/AML3白血病细胞株以及对照白血病细胞株HL60。采用q RT-PCR、Western blot和免疫细胞化学法检测白血病细胞的sh NPM组以及Mock和p GIPZ组细胞及NPM1 A型突变(NPM1-m A)基因和蛋白的表达,流式细胞仪和瑞氏染色法观察OCI/AML3的sh NPM组以及Mock和p GIPZ组细胞凋亡情况,q RT-PCR和Western blot检测OCI/AML3的sh NPM组以及Mock和p GIPZ组凋亡相关蛋白(Bax/Bcl-2)和p-ERK信号分子表达;流式细胞仪检测丝裂原活化蛋白激酶/胞外信号调节激酶信号通路抑制剂(PD98059)处理后OCI/AML3的sh NPM组以及Mock和p GIPZ组细胞凋亡率的改变。结果:干扰NPM1明显抑制OCI/AML3细胞株NPM1-m A的m RNA水平(F=20.078;P=0.000,PMock vs.sh NPM=0.001,Pp GIPZ vs.sh NPM=0.003,PMock vs.p GIPZ=0.333)和蛋白水平,伴有胞质NPM突变蛋白表达明显减弱;干扰NPM1能明显上调OCI/AML3细胞凋亡率(F=25.236,P=0.000,PMock vs.sh NPM=0.001,Pp GIPZ vs.sh NPM=0.001,PMock vs.p GIPZ=0.729),同时光镜下观察到干扰组细胞出现凋亡小体等凋亡形态学特征。此外,干扰NPM1可上调OCI/AML3细胞中促凋亡分子Bax的蛋白和m RNA表达(F=7.649,P=0.000;PMock vs.sh NPM=0.015,Pp GIPZ vs.sh NPM=0.015,PMock vs.p GIPZ=0.984)、下调抗凋亡分子Bcl-2的蛋白和m RNA表达(F=5.190,P=0.000;PMock vs.sh NPM=0.034,Pp GIPZ vs.sh NPM=0.029,PMock vs.p GIPZ=0.909);干扰NPM1能明显下调p-ERK的表达,PD98059抑制剂阻断MAPK通路可促进OCI/AML3细胞凋亡(F=25.643,P=0.007)。结论:NPM1突变基因在白血病细胞抗凋亡特性中发挥重要作用,潜在的分子机制可能与ERK信号分子及Bax/Bcl-2表达有关。Objective：To investigate the effect of nucleophosmin 1 （NPM1） on the apoptosis in leukemic cells and the relevant molecular mechanisms. Methods：OCI/AML3 cell lines（NPM1 mutant type） and HL60 cell lines（NPM1 wild type） were served as experimental group and negative control group. Mock group,pGIPZ group,shNPM group were established in the meantime. Expressions of the NPM 1 mutant A（NPM 1-mA） gene and protein were detected by qRT-PCR, Western blot and immune cytochemistry. Apoptosis analysis was determined by flow cytometry and Wright-Giemsa staining. The gene and protein expression of apoptosis related molecular （Bax/Bcl-2） was detected by qRT-PCR and Western blot and the activity of p-ERK was also assessed by Western blot. The percent- age of apoptotic cells was detected after treatment with MAPK/ERK pathway inhibitor（PD98059） by flow cytometry. Results：The results showed significant down-regulation of NPM1-mA mRNA levels（F=20.078,P=0.000;PMock vs.shNPM=0.001 ,PpGIPZ vs. shNPM=0.003,PMock vs.pGIPZ=0.333） and protein levels after NPM1 silencing, accompanying by reduce of cytoplasm NPM mutant protein of OCI/AML3 cells. In OCI/AML3 ceils, down-regulation of NPM1-mA resulted in increase of cell apoptofic rate（F=25.236,P=0.000;PMock vs.shNPM=0.001, PpGIPZ vs. shNPM=0.001 ,PMock vs.pGIPZ=0.729） and morphological changes including apoptosis bodies under light microscopy. NPM1 gene si-lencing led to increase of pro-apoptotic protein Bax expression （F=7.649,P=0.000; PMock vs.shNPM=0.015,PpGIPZ vs. shNPM=0.015, PMock vs. shNPM= 0.984） and decrease of anti-apoptotic protein Bcl-2 expression at mRNA and protein levels（F=5.190,P=0.000,PMock vs.shNPM=0.034, PpCIPZ vs shNPM=0.029,PMock vs pGIPZ=0.909）. NPM1 gene silencing can also down-regulate the expression of p-ERK. Furthermore,PD98059 promoted OCI/AML3 leukemic cell apoptosis（F=25.643,P=0.007）. Conclusion ： NPM1-mA plays an important role in leukemic cell anti-apoptosis, which involving MAPK signal pathway and Bax/Bcl-2 exp

关 键 词：白血病 核仁磷酸蛋白 基因突变 OCI/AML3细胞 凋亡 

分 类 号：R733.71[医药卫生—肿瘤]