重组腺相关病毒协同重组腺病毒增强目的基因表达的体内试验

摘要

Objective To study the changes in the intensity and temporal pattern of target gene expression in the tumor tissue of nude mice bearing human nasopharyngeal carcinoma (NPC) following injection of recombinant adeno-associated virus (rAAV) and recombinant adenovirus (AdV) in vivo. Methods EBV-positive human NPC cell line C666-1 was inoculated subcutaneouly in nude mice. After the tumor mass reached 3 mm in diameter, 1.5x1011 v.g (virus genome) rAAV-EGFP, 2.5x10" pfu rAdV-EGFP or their balanced mixture was injected intratumorally. At 5 and 10 days after the injection, the tumor tissues were harvested for immunohistochemical staining of GFP, and the ratio of the GFP-positive cells and the intensity of GFP expression was determined. Results Immunohistochemistry for GFP showed that 5 days after the injection, GFP expression was detected (1.70±0.48) in the tumor tissue in rAAV group, and the peak expression levels was seen in rAdV group (6.00±1.94); the expression level was comparable between the combination group (6.90±1.92) and rAdV group. At 10 days, GFP expression was considerably lowered to 2.00±0.67 in rAdV group but increased to 8.00±1.15 in rAAV group. The expression in the combination group maintained a high level at 10 days (10.10±1.63), which was significantly higher than that in rAAV group (P=0.001). Conclusion Transfection with rAAV combined with rAdV allows instant, sustained and significantly enhanced expression of the target gene in the tumor tissue. This approach takes advantages of the two viruses and can be ideal for exogenous gene delivery into the tumor tissues.%目的 研究重组腺相关病毒(rAAV)协同重组腺病毒(AdV)在体内增强目的 基因表达的强度及时间变化.方法 人EB病毒阳性鼻咽癌细胞株C666-1接种于裸鼠皮下,待肿瘤直径达到3 mm左右以rAAV-EGFP 1.5×1011v.g(病毒基因组数,virus genome)、rAdV-EGFP2.5×108pfu或其等量混合液采用瘤内注射,于注射后第5天、10天分别处死裸鼠,取注射点周围肿瘤组织以10%PBS福尔马林固定,用免疫组化方法检测GFP表达情况,在400×光学显微镜下计数具有代表性的10个视野下的阳性细胞比例及强度,采用SPSS10.0单项方差分析统计结果.结果 免疫组化提示瘤内注射病毒后第5天rAAV组开始表达目的 基因(1.70±0.48),rAdV组目的 基因表达达到高峰(6.00±1.94),联合组表达量与AdV组基本一致(6.90±1.92);第10天rAdV组目的 基因表达基本消失(2.00±0.67),rAAV组表达明显升高(8.00±1.15),联合组持续高表达(10.10±1.63),且较rAAV组有显著差异(P=0.001).结论 rAAV协同rAdV能够显著提高目的 基因的表达量,能够使目的 基因表达迅速且维持较长时间,可相互弥补两种病毒的不足,是一种值得推荐的基因表达方式.