慢病毒介导的siRNA靶向CD47基因对人喉癌细胞Hep-2增殖、凋亡的影响  

Effect of small interfering RNA targeting CD47 gene mediated by lentivirus vectors on proliferation and apoptosis of human laryngocarcinoma Hep-2 cells

作　　者：高树峰[1] 张少容[1] 徐莲[1] 李黎[1] 万俊[1] 杨春平[1] 刘建国[1] 汪美群[1] 刘月辉[1] 

机构地区：[1]南昌大学第二附属医院耳鼻咽喉头颈外科,南昌330006

出　　处：《解放军医学杂志》2013年第8期634-638,共5页Medical Journal of Chinese People's Liberation Army

基　　金：江西省自然科学基金(20114BAB205069)~~

摘　　要：目的探究慢病毒载体介导siRNA抑制CD47基因表达后对人喉癌细胞株Hep-2体外增殖凋亡的影响。方法构建慢病毒载体,将靶向CD47基因的siRNA慢病毒转染至Hep-2细胞;用荧光显微镜行细胞形态学变化观察;RT-PCR检测细胞CD47 mRNA表达的变化;Western blotting检测CD47蛋白表达的变化情况;MTT法检测细胞的增殖凋亡情况。结果 CD47-siRNA慢病毒转染Hep-2细胞后,形态学观察显示CD47-siRNA能有效抑制Hep-2细胞增殖,细胞变形明显,体积变小,可见细胞坏死及凋亡。RT-PCR和Western blotting检测显示,CD47的mRNA和蛋白表达水平显著降低（P〈0.05）,使CD47 mRNA表达减少76%~82%,CD47蛋白表达减少77%;慢病毒转染细胞在48h后MTT检测细胞凋亡率明显提高（P〈0.01）。结论慢病毒介导的siRNA干扰CD47基因表达后,明显抑制了喉癌Hep-2细胞CD47的表达并且诱导了Hep-2凋亡。Objective To study the effect of small interfering RNA（siRNA） targeting CD47 gene mediated by lentivirus vectors on proliferation and apoptosis of human laryngocarcinoma Hep-2 cells.Methods After building lentivirus vector,a siRNA directed against CD47 gene was transfected into Hep-2 cells.Cell morphological changes were observed by fluorescence microscopy.The changes in CD47 mRNA expression were detected by semi-quantitative RT-PCR,and the protein of CD47 was evaluated by Western blotting.Suppression of proliferation and apoptosis of Hep-2 cells were observed by MTT assay.Results After the laryngocarcinoma Hep-2 cells were transfected by CD47-siRNA lentiviral plasmid,it was showed by fluorescence microscopy that the CD47 siRNA was able to effectively suppress the Hep-2 cell proliferation,the cells were deformed and diminished in size obviously,and necrosis and apoptosis were observed.Semi-quantitative RT-PCR and Western blotting revealed that the expression of CD47 mRNA and protein decreased by 76%–82% and 77%,respectively（P＆lt;0.05）.Forty-eight hours ater the lentivirus vectors transfection,MTT assay revealed that cell apoptosis increased significantly（P＆lt;0.01）.Conclusion Small interfering RNA targeting CD47 gene mediated by lentivirus vectors can signiicantly inhibit CD47 gene expression in Hep-2 cells and induce cell apoptosis in vitro.

关 键 词：CD47 慢病毒载体 RNA干扰 HEP-2细胞 基因沉默 

分 类 号：R739.65[医药卫生—肿瘤]