EB病毒潜伏膜蛋白1对鼻咽癌细胞体外转移能力影响  

Effect of Epstein-Barr Virus-encoded latent membrane protein 1 on the metastasis ability of nasopharyngeal cells in vitro

作　　者：刘雄[1] 彭英[2,3] 李刚[1] 张宝[3] 李晓华[4] 周立辉[1] 李湘平[1] 

机构地区：[1]南方医科大学南方医院耳鼻咽喉头颈外科,广东广州510515 [2]中山大学第二附属医院神经内科,广东广州510120 [3]南方医科大学分子生物教研室,广东广州510515 [4]解放军第九四医院耳鼻咽喉科,江西南昌330002

出　　处：《中国耳鼻咽喉头颈外科》2008年第11期615-619,共5页Chinese Archives of Otolaryngology-Head and Neck Surgery

基　　金：国家自然科学基金项目(30471945);广东省科技计划攻关项目(2D03C30303)联合资助

摘　　要：目的探讨EB病毒（epstein-barr virus,EBV）潜伏膜蛋白1（Iatent membrane protein1，LMP-1）对鼻咽癌（nasopharyngeal carcinoma，NPC）细胞体外转移能力的影响。方法设计针对EBV-LMP-1的特异性发夹状RNA（small hairpin RNA，shRNA）干扰序列，构建2种重组腺相关病毒（recombinant adeno—associated virus，rAAV）载体：rAAV-增强型绿色荧光蛋白（enhance green fluorescent protein，EGFP）和rAAV-shRNA—LMP-1，以不同滴度rAAV-EGFP转染鼻咽癌C666—1细胞确定最佳转染效率（multiplicity of infection，MOI），rAAV-shRNA-LMP-1按MOI转染C666-1，RT-PCR鉴定抑制效率，划痕试验和基底膜穿透试验检验细胞转移能力的变化。结果以rAAV-EGFP5×10^4v．g（virus genome，病毒基因组数），细胞转染C666—1细胞，转染效率大于95％，RT-PCR鉴定rAAV-shRNA—LMP-1以5×10^4v．g／细胞转染C666-1后目的基因抑制效率大于90％，划痕试验显示在相同的时间内，越过划痕边缘移动到空白处的细胞数明显减少（P〈0．01），基底膜穿透试验提示细胞穿透能力显著下降（P〈0．001）。结论通过rAAV介导RNA干扰能有效抑制LMP-1基因表达，并显著抑制了肿瘤细胞的运动及穿透转移能力。OBJECTIVE To study the effect of Epstein-Barr virus（EBV） encoded latent membrane protein I（LMP-1） on the metastasis potential of nasopharyngeal carcinoma（NPC） cells in vitro. METHODS Design specific interference small hairpin RNA（shRNA） on EBV-LMP-1 and construct two recombinant adeno-associated virus （rAAV）： rAAV-shRNA-LMP-1 and rAAV-EGFP （enhance green fluorescent protein）. Multiplicity of infection （MOI） was confirmed by using different titer of rAAV-EGFP to transfect NPC cell line, C666-1. Then C666-1 cells were transfected by rAAV-shRNA-LMP-1 at MOI titer and the inhibiting efficiency of target gene＇s expression was confirmed by reverse transcription polymerase chain reaction （RT-PCR）. The metastasis potential of NPC cells was confirmed by wound healing assay and in vitro Matrigel invasion assay. RESULTS The transfection efficiency was exceeded 95 % with 5×10^4 virus genome（v.g）/cell rAAV-EGFP. The expression of target gene was inhibited over 90 % assessed by RTPCR after transfected with rAAV-shRNA-LMP-1 at 5x 104 v.g/cell. The cell number that transmigrated to the blank space was significantly lower than control group at the same period （P 〈0.01） and the rAAV-shRNA-LMP-1 transfected cells displayed a significantly lower transmembrane migration activity （P 〈0.001）. CONCLUSION The expression of LMP-1 can be suppressed effectively by rAAV mediated RNA interference. The ability of cell migration and cell invasiveness can be inhibited significantly after LMP-1 was suppressed.

关 键 词：鼻咽肿瘤 依赖病毒 RNA干扰 肿瘤转移 潜伏膜蛋白1 

分 类 号：R739.63[医药卫生—肿瘤]