重组腺相关病毒介导RNA干扰抑制EB病毒潜伏膜蛋白1对鼻咽癌细胞影响的动物试验  

Effect of rAAV-mediated RNA interference against EB virus-encoded latent membrane protein 1 on nasopharyngeal carcinoma cell proliferation and metastasis in nude mice

作　　者：刘雄[1] 李刚[1] 张宝 王路[1] 李晓华[3] 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 [2]分子生物教研室,广东广州510515 [3]江西南昌解放军第九四医院耳鼻喉科,江西南昌330002

出　　处：《南方医科大学学报》2009年第4期611-614,618,共5页Journal of Southern Medical University

基　　金：国家自然科学基金项目(30471945);广东省科技计划攻关项目(2003C30303)

摘　　要：目的探讨EB病毒(EBV)潜伏膜蛋白1(LMP-1)对于鼻咽癌细胞在体内增殖及转移能力的影响。方法设计针对EBV-LMP-1的特异性发夹状RNA(shRNA)干扰序列,构建2种重组腺相关病毒(rAAV)载体:(增强型绿色荧光蛋白,EGFP)rAAV-EGFP和rAAV-shRNA-LMP-1,以不同滴度rAAV-EGFP转染鼻咽癌C666-1细胞确定最佳转染复数(MOI),rAAV-shRNA-LMP-1按MOI转染C666-1,RT-PCR鉴定抑制效率,将体外转染RNA干扰后的C666-1细胞注入裸鼠肝脏包膜下制作鼻咽癌异位肝种植的肝肺转移模型,观察肝脏成瘤及肝内、肺转移情况,分析LMP-1基因沉默在动物水平对鼻咽癌细胞成瘤及转移能力的影响。结果rAAV-EGFP以5×104v.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5×104v.g/细胞转染C666-1后目的基因抑制效率大于90%,动物试验结果显示rAAV-shRNA-LMP-1组肝脏成瘤体积与对照组rAAV-EGFP无显著差异,但显著减少了种植肿瘤的肝内及肺转移率。结论通过rAAV介导RNA干扰能有效抑制LMP-1基因表达,可在动物水平抑制鼻咽癌细胞转移,但对鼻咽癌细胞生长无显著影响。Objective To study the role of Epstein-Barr virus （EBV）-encoded latent membrane protein 1 （LMP-1） in nasopharyngeal carcinoma （NPC） cell growth and metastasis in vivo by recombinant adeno-associated virus （rAAV）-mediated RNA interference （RNAi）. Methods Specific small hairpin RNA （shRNA） targeting EBV-LMP-1 gene was designed and synthesized to construct two rAAV vectors rAAV-shRNA-LMP-1 and rAAV-EGFP. The multiplicity of infection （MOI） was confirmed using different titers ofrAAV-EGFP to transfect the NPC cell line C666-1. The C666-1 cells were transfected by rAAV-shRNA-LMP-1 at the optimal MOI titer and the inhibition efficiency of the target gene expression was determined with RT-PCR. The C666-1 cells with RNAi of LMP-1 gene were injected into nude mouse liver via laparotomy to establish the animal model of hepatic and lung metastases of NPC cells. The metastases of the C666-1 cells in the liver and lungs were observed to assess the effect ofLMP-1 gene silencing on the tumorigenic and metastatic potentials of the cells in vivo. Results The transfection efficiency of 5 x 104 virus genome/cell rAAV-EGFP exceeded 95%. The expression of the target gene was suppressed by over 90% as shown by RT-PCR after transfection with rAAV-shRNA-LMP-1 at 5 ×10^4 virus genome/cell. Animal experiments showed that compared with rAAV-EGFP, rAAV-shRNA-LMP-1 transfection did not reduce the primary tumor volume implanted into the liver, but significantly inhibited the intrahepatic and lung metastases of the NPC cells. Conclusion LMP-1 expression can be suppressed effectively by rAAV-mediated RNAi, and LMP-1 suppression does not obviously affect the tumor cell growth but can inhibit their metastasis in vivo.

关 键 词：鼻咽癌 重组腺相关病毒 RNA干扰 EB病毒潜伏膜蛋白1 转移 

分 类 号：R739.63[医药卫生—肿瘤]