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.
出处
《中国耳鼻咽喉头颈外科》
北大核心
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
关键词
鼻咽肿瘤
依赖病毒
RNA干扰
肿瘤转移
潜伏膜蛋白1
Nasopharyngeal Neoplasms
Adeno-Associated Viruses
RNA Interference
NeoplasmMetastasis
latent membrane protein 1
作者简介
第一作者简介及通讯：刘雄，男，云南人，医学硕士，主治医师，主要从事头颈肿瘤临床与基础研究。Email：liux1218@126.com..
通讯作者：李湘平（Email：lp133402@yahoo．com．cn）
