EBV-LMP1对鼻咽癌细胞系CNE1细胞凋亡的影响

背景与目的:已证实EB病毒编码的潜伏膜蛋白1(latentmembraneprotein,LMP1)具有转化致瘤作用,在鼻咽癌的发生发展中起重要作用。LMP1的转化致瘤作用可能与细胞凋亡有关,本实验目的是观察EBV-LMP1对鼻咽癌细胞系CNE1细胞凋亡的影响,探索LMP1在鼻咽癌发生发展中的可能机制。方法:用电转染的方法将带有绿色荧光蛋白GFP报道系统的EB病毒LMP1基因真核表达质粒导入CNE1,用荧光显微镜检测报道基因GFP的表达情况;用免疫组化法及Westernblot法检测目的基因LMP1的表达情况;用流式细胞仪、荧光染色及DNA片段分析等方法检测在有地塞米松磷酸钠或无血清饥饿诱导的情况下LMP1对CNE1细胞凋亡的影响。结果:CNE1G细胞(转染空载质粒PAT-GFP的CNE1细胞)及CNE1GL(转染目的基因质粒PAT-GFP-LMP1的CNE1细胞)有绿色荧光蛋白表达,CNE1细胞(未经转染的CNE1细胞)无绿色荧光蛋白表达;CNE1GL细胞LMP1呈阳性表达,而CNE1及CNE1G细胞均为阴性。CNE1、CNE1G及CNE1GL3组细胞分别用地塞米松磷酸钠或无血清1640培养液处理后均有凋亡出现,且两种诱导方法均能使CNE1GL组细胞的凋亡指数(apoptosisindex,AI)较CNE1及CNE1G组明显增高(P<0.05),而CNE1与CNE1G组的凋亡指数无明显差别。3组细胞常规培养下则无凋亡出现。结论:LMP1基因成功导入CNE1细胞

Effect of Epstein-Barr virus latent membrane protein 1 (LMP1) on apoptosis of nasopharyngeal carcinoma cell line CNE1

Background &Objective:It was proved that Epstein-Brarr vir us(EBV)latent membrane protein 1(EBV-LMP1)has transformation function which p lays an important role in the genesis and development of nasopharyngeal carcinoma (NPC)and this function may be associated w ith apoptosis.This study was design ed to investigate the effect of EBV-LMP1on apoptosis of NPC CNE1cell line and to explore the possible mec hanism of the function of EBV-LMP1in the genesis and development of NPC.Methods:The authors transfected LMP1eukary otic expression plasmid carried wit h the green fluorescent protein(GFP)reporter gene into CNE1cell line by e lectroporation.The expression of G FP was detected by fluorescent microscope;the expression of aimed gene LMP1was detected b y immunohistochemistry and Western blot;the effect of LMP1on apoptosis of CNE1cells which were treated either with dexamethasone sodium phosphate or serum starvation was investigated by flow cytometry analysis,fluorescence s taining ,and DNA fragmentation analysis.Results:The green fluorescence could be detecte d in CNE1G cells transfected by contr ol plasmid PAT-GFP and in CNE1GL cell s transfected by plasmid PAT-GFP-LMP 1,while there was no GFP expression i n CNE1cells untransfected by any pla smid.LMP1could be detected in CNE1GL cell s,but not in CNE1and CNE1G cells.Apoptosis could be obse rved in three groups cells which were treated either with dexam ethasone sodium phosphate or serum starvation,and the apoptosis index(AI )of CNE1GL cells 499was higher than that in CNE1and CNE1G cells(P<0.05).There were no apoptosis occurring i n three group cells when cultured in regular medium.Conclusions:The LMP1gene was transfected successfully into CNE1cells which were stably expressing LMP1.LMP1could i ncrease apoptosis occurring of CNE1cells in the process of inducing apop tosis.