重组慢病毒LV5-NE的构建及NE促进NB4细胞增殖  

Construction of recombinant lentiviral vector and neutrophil elastase promotes proliferation in NB4 cell

作　　者：杨蓉[1,2] 钟梁[2] 刘北忠[1,2] 阳小群[2] 蒋开玲[2] 李浏[2] 宋浩[2] 

机构地区：[1]重庆医科大学附属永川医院中心实验室,重庆402160 [2]重庆医科大学临床检验诊断学教育部重点实验室,重庆400016

出　　处：《基础医学与临床》2016年第1期35-40,共6页Basic and Clinical Medicine

基　　金：国家自然科学基金(81171658);重庆市自然科学基金重点项目(2011BA5037)

摘　　要：目的构建重组慢病毒LV5-NE筛选纯NB4/LV5-NE细胞株,探讨中性粒细胞弹性蛋白酶(NE)对人急性早幼粒细胞白血病细胞株NB4增殖的影响,为研究NE致白血病的可能机制奠定基础。方法以质粒p CMV-myc-NE为模板得到PCR产物NE,将NE基因连接到穿梭载体p GLV10/U6/GFP/Puro后酶切验证,将序列正确的质粒与包装质粒p Gag/Pol、pRev、p VSV-G共转染293T细胞。测定病毒滴度。实验分为空白组、LV5-NC组和LV5-NE组,病毒感染NB4细胞72 h后用嘌呤霉素筛选稳定表达的细胞。用Western blot检测NE在NB4细胞中的蛋白表达以及其切割作用;CCK-8法观察NB4细胞增殖能力;流式细胞术测定NB4细胞周期和凋亡;Western blot检测p Akt和Akt以及Bax和Bcl-2蛋白表达。结果成功构建表达NE的慢病毒LV5-NE,筛选得到纯NB4/LV5-NE细胞株,NE对NB4细胞的增殖有明显促进作用(P<0.05)结论 NE在NB4细胞株中稳定表达后中可促进NB4细胞的增殖,其增殖作用可能通过激活PI3K/Akt通路调节。Objective To construct recombinant lentiviral LV5-NE and screen NB4 / LV5-NE cell line. To explore the effects of neutrophil elastase on proliferation and apoptosis in NB4 cell. Methods Calculating the title of LV5-NE by fluorescence counting. There were three groups（ blank control group,LV5-NC group,LV5-NE）,Western blot was used to detect the expression of NE protein and its cleaved products in NB4 cell after infection by LV5-NE. CCK-8 was used for cell proliferation analysis. Flow cytometry was used for cell cycle and apoptosis analysis.Western blot was used to detect the expression level of phosphorylated Akt,Akt,Bax and Bcl-2 in NB4 cell.Results The lentivir of LV5-NE and screen NB4 / LV5-NE cells were constructed. Neutrophil elastase obviouslypromoted proliferation and inhibited apoptosis in NB4 cell（ P〈0. 05）. Conclusions Neutrophil elastase may active PI3 K / Akt pathway to promote proliferation and inhibit apoptosis in NB4 cell.

关 键 词：中性粒细胞弹性蛋白酶 慢病毒构建 增殖 凋亡 NB4细胞 

分 类 号：R733.71[医药卫生—肿瘤]