摘要目的 探讨EB病毒(EBV)潜伏膜蛋白1(LMP-1)对鼻咽癌细胞在体内增殖和转移能力的影响及其可能的机制.方法 构建重组腺相关病毒(Raav)-增强型绿色荧光蛋白(EGFP)和Raav-shRNA-LMP-1载体.以不同滴度的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细胞,采用逆转录聚合酶链反应(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建立鼻咽痛移植瘤模型,观察裸鼠的肝脏成瘤及肝脏、肺脏转移情况.采用免疫组化法检测基质金属蛋白酶9(MMP-9)的表达,分析LMP-1基因沉默对鼻咽癌细胞成瘤和转移能力的影响及机制.结果 Raav-EGFP以5×104病毒基因组数/细胞转染C666-1细胞,转染效率>95%.Raav-shRNA-LMP-1以5×104病毒基因组数/细胞转染C666-1后,LMP-1的表达抑制率>90%.Raav-shRNA-LMP-1转染组裸鼠的肝脏成瘤体积为(0.2527±0.1152)cm3,与Raav-EGFP转染组[(0.2533±0.0754)cm3]的差异无统计学意义(P>0.05),但Raav-sbRNA-LMP-1转染组裸鼠的肝转移率(50.0%)和肺转移率(33.3%)明显低于Raav-EGFP转染组(均P<0.05).Raav-shRNA-LMP-1转染组裸鼠的生存时间为(15.50±2.47)d,明显长于Raav-EGFP转染组[(11.50±1.22)d,P<0.05].免疫组化染色结果 显示,LMP-1抑制后,MMP-9的表达明显下调.结论 通过Raav介导RNA干扰序列能有效抑制LMP-1的表达,其可能通过下调MMP-9的表达抑制鼻咽癌细胞的转移,但对鼻咽癌细胞的生长无显著影响.

abstractsObjective To study the effect of Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP-1) on the tumor growth and metastasis of nasopharyngeal carcinoma (NPC) cells in vivo and its possible mechanism. Methods To construct two recombinant adeno-associated virus (rAAV) : rAAV-shRNA-LMP-1 and rAAV-EGFP (enhanced green fluorescent protein). Multiplicity of infection (MOI) was confirmed by using different titre of rAAV-EGFP to transfect a NPC cell line, C666-1. Then C666-1 cells were transfected by rAAV-shRNA-LMP-1 at MOI titre and the inhibiting efficiency of target gene's expression was confirmed by reverse transcription polymerase chain reaction (RT-PCR). C666-1 cells treated by RNAi on LMP-1 in vitro was directly inoculated into the liver via laparotomy under direct vision to establish the animal model of NPC xenograft in liver and lung metastasis from the liver. The biological effect and its mechanism after "gene silencing" of LMP-1 on NPC cells tumorigenesis and metastasis were observed by the primary intrahepatic tumor formation and lung metastasis and the expression of matrix metalloproteinases-9 (MMP-9) revealed by immunohistochemistry. Results The transfection efficiency was higher than 95% with 5 × 104 virus genome (v.g) /cell with rAAV-EGFP. The expression of target gene was inhibited more than 90%, assessed by RT-PCR after transfection with rAAV-shRNA-LMP-1 at a dose of 5 × 104 v. G/cell. The primary tumor volume implanted in the liver of rAAV-shRNA-LMP-1 treatment was (0.2527 ± 0.1152) cm3, with no significant difference in comparrison with rAAV-EGFP control group [(0. 2533 ± 0. 0754) cm3, P >0.05]. But the rate of intrahepatic tumor formation was 50.0% and the rate of lung metastasis was 33.3% of the rAAV-shRNA-LMP-1 group, significantly lower than those in the rAAV-EGFP group (P < 0.05). The survival time(15.50 ± 2.47) d of rAAV-shRNA-LMP-1 group was significantly longer than that in the rAAV-EGFP group [(11.50 ± 1.22) d, P <0. 05]. Immunohistochemistry indicated suppression of LMP-1 by rAAV-shRNA-LMP-1 can down-regulate the expression of MMP-9 significantly. Conclusion The expression of LMP-1 can be suppressed effectively by rAAV mediated RNA interference. The suppression of LMP-1 expression has no effect on cell growth but can inhibit the metastasis in vivo, probably through down-regulating the expression of MMP-9.