利用腺病毒提高腺相关病毒对肿瘤细胞的转导效率

Enhanced transduction efficiency of adeno-associated virus on cancer cells with the help of low dose adenovirus

目的探讨小量腺病毒作为辅助病毒能否增强腺相关病毒(AAV)对肿瘤细胞和裸鼠肿瘤模型的转导效率并对增强的机理进行初步探讨。方法采用绿色荧光蛋白(EGFP)标记的AAV2联合不同滴度的非复制型腺病毒(Ad-null)感染人非小细胞肺癌细胞系(AAV2+Ad-null 组),并以相同剂量的 AAV2单独感染(AAV2组)作为对照。用荧光显微镜观察 EGFP 阳性细胞,流式细胞仪检测 EGFP 阳性细胞的百分率和单个细胞平均荧光强度,Western 印迹检测 EGFP 蛋白表达的变化以比较各组病毒转导效率。换用萤光素酶(Luc)标记的 AAV2,通过 Luc 检测系统检测两组对体外肿瘤细胞及裸鼠肿瘤模型的转导效率。荧光定量 PCR 检测感染后肿瘤细胞 DNA 及 mRNA 的 EGFP相对拷贝数,观察 Ad-null 对 AAV2复制及转录的影响。结果随着 Ad-null 滴度的增加,AAV2+Ad-null组的肿瘤细胞 EGFP 阳性细胞数明显增加。流式细胞仪计数的 EGFP 阳性细胞率按0.01、0.1、1、10 MOI Ad-null 滴度依次为10.9%,18.0%,36.2%,55.2%,明显高于 AAV2组(6.4%)。AAV2+10 MOI Ad-null 组单个细胞的荧光强度约为 AAV2组的1.32倍。各 AAV2+Ad-null 组 EGFP在细胞中的蛋白表达水平也均高于 AAV2组。体外、体内Luc检测均显示 Ad-null 显著增强 AAV2对肿瘤的转导效率,荧光素信号分别为 AAV2单独感染的28和4.5倍。Ad-null 可提高肿瘤细胞内AAV2的 mRNA 水平,当 Ad-null 滴度为1、10 MOI 时 AAV2+Ad-null 组的 EGFP 拷贝数明显高于AAV2组,而 Ad-null 对细胞内 AAV2的 DNA 影响不大,AAV2+Ad-null 组与 AAV2组的 EGFP 拷贝数差别不大。结论联合使用少量腺病毒可明显提高 AAV 对肿瘤细胞的转导效率,可能与其提高AAV 转录水平有关,为今后应用 AAV 作为肿瘤基因治疗的载体提供了实验证据。

Objective To investigate whether adeno-associated virus （AAV） could enhance its infection efficiency on cancer cells when combined with non-replicable adenovirus （Ad-null） in vitro as well as in vivo and to study its underlying mechanisms. Methods AAV2 particle was added into NCI-H460 tumor cell lines alone or in combination with different amount of adenovirus. 1 to 7 days after transduction, cells were observed and recorded with fluorescence microscope, the expression levels of report gene EGFP in tumor cells were examined by using flow cytometry and Western blotting, the expression of report genes luciferase was analyzed with luminometer to obtain the relative light units. After the establishment of tumor model, the nude mouses were administrated with AAV2 or AAV2 ＋ Ad-null in tumors, and then tested their infection efficiency and expression levels with roperscientific bioluminescence tumor imaging system. Results The results obtained with the help of flow cytometry and luciferase assay suggested that the infection efficiency of AAV2 was enhanced significantly when combined with low dose Ad-null in vitro, the infection efficiency of AAV2 alone was 6.4% and it reached 55.2% when combined with 10 MOI Ad-null, Western blotting assay demonstrated that the protein expression level of reporter gene in tumor cells enhanced when combined with 10 MOI Ad-null compared with AAV2 infection alone, and the enhancement of reporter gene expression was observed in a concentration-dependent manner; real-time PCR analysis confirmed that Ad- null enhanced the mRNA level of AAV2-EGFP but not the copies of genomic DNA of AAV2-EGFP. Ad-null significantly augmented the infection efficiency when tested on NCI-H460 tumor model. With the help of Ad- null, the signal of luciferin in nude mouse was 4. 5 times more than that of control. Conclusion The infection efficiency of AAV was enhanced significantly when combined with low dose Ad-null in vitro and in vivo, and it offers basis for further study of gene therapy by AAV.