LMP1诱导鼻咽癌细胞发生TRAIL抵抗的体外实验  

In vitro study of LMP1-induced TRAIL resistance in nasopharyngeal carcinoma cells

作　　者：李京鲲 尹丹辉 王爽[1] 李平[1] 杨新明[1] 李仕晟[1] LI Jing-kun;YIN Dan-hui;WANG Shuang;LI Ping;YANG Xin-ming;LI Shi-sheng(Department of Otolaryngology-Hend and Neck Surgery, the Second Xiangya Hospital, Central South University, Changsha 410011, China)

机构地区：[1]中南大学湘雅二医院耳鼻咽喉头颈外科,湖南长沙410011

出　　处：《中国耳鼻咽喉颅底外科杂志》2018年第3期219-225,共7页Chinese Journal of Otorhinolaryngology-skull Base Surgery

基　　金：国家自然科学基金(81402502)

摘　　要：目的探讨EBV膜潜伏蛋白1(latent membrane protein 1,LMP1)基因过表达后对肿瘤坏死因子相关的凋亡诱导配体(TNF-related apoptosis-inducing ligand,TRAIL)凋亡诱导作用和凋亡信号传导的影响。方法利用脂质体介导的pGL6-LMP1上调LMP1低表达的TRAIL抵抗性鼻咽癌细胞CNE-1中LMP1的表达;通过MTT比色法、流式细胞术观察LMP1过表达后对CNE-1细胞TRAIL敏感性的影响;流式细胞术、Western blot、线粒体膜电位检测观察LMP1过表达后对死亡受体表达、细胞内外凋亡信号通路激活、线粒体膜电位改变的影响。结果死亡受体荧光标记后流式细胞术检测发现CNE-1和CNE-1-LMP1之间细胞膜蛋白死亡受体4(death receptor 4,DR4),死亡受体5(death receptor,DR5)表达差异无统计学意义(P>0.05)。Western blot结果显示TRAIL(100 ng/ml)分别作用2、4、6、12 h后,CNE-1细胞中caspase-8 p43/p41,p18亚单位蛋白和caspase-3 p17,p10亚单位蛋白表达明显高于CNE-1-LMP1细胞,而Bcl-2相互作用域死亡激动蛋白的截短形式(truncated form of Bid,t Bid)和caspase-9 p35亚单位蛋白表达无明显改变,且两个细胞株间也无明显区别。JC-1线粒体膜电位检测法结果显示TRAIL干预后,线粒体膜电位无明显改变,且两个细胞株间也无明显区别。结论 LMP1过表达抑制TRAIL对鼻咽癌细胞的凋亡诱导作用和其细胞外凋亡信号的传导,提示LMP1是通过抑制细胞外信号通路激活诱导鼻咽癌细胞发生TRAIL抵抗。ObjectiveTo observe the effect of latent membrane protein 1 （LMP1） over-expression on TNF-related apoptosis-inducing ligand（TRAIL）-induced apoptosis and TRAIL-activated apoptotic pathway.MethodsLMP1 expression in CNE-1 which was TRAIL-resistant LMP1 low expressive nasopharyngeal carcinoma （NPC） cell was up-regulated by liposome mediated pGL6-LMP1. The TRAIL sensitivities of CNE-1-LMP1 and CNE-1 were measured by MTT and flow cytometry. The expression of death receptor （DR）, activation of apoptotic pathway and mitochondrial transmembrane potential were detected by flow cytometry, Western blot and JC-1 assessment, respectively.ResultsFlow cytometry showed no significant differences in expressions of DR4 and DR5 between CNE-1 and CNE-1-LMP1 cell lines （all P〉0.05）. Moreover, after TRAIL （100 ng/ml） administration for 2, 4, 6, 12 hours, the expressions of caspase-8 p43/p41, tathmin/oncoprotein 18p18 and caspase-3 p17, p10 in CNE-1 were higher than those in CNE-1-LMP1. However, there were no significant differences in expressions of tBid and caspase-9 p35 between this two cell lines. JC-1 assessment showed that TRAIL administration could not affect the mitochondrial transmembrane potential and there was no significant difference in mitochondrial transmembrane potential between CNE-1 and CNE-1-LMP1.ConclusionLMP1 over-expression can inhibit apoptosis and extracellular apoptotic pathway, which indicates LMP1 may induce TRAIL-resistance in NPC cell through inhibiting extracellular apoptotic pathway.

关 键 词：LMP1基因 TRAIL 凋亡 抵抗 

分 类 号：R739.63[医药卫生—肿瘤]