论文部分内容阅读
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 the small amount of adenovirus as a helper virus can enhance the transduction efficiency of adeno-associated virus (AAV) on tumor cells and nude mice tumor model and to explore the mechanism of its enhancement. Methods Human non-small cell lung cancer cell line (AAV2 + Ad-null) was infected with green fluorescent protein (EGFP) -added AAV2 and different titer of non-replicating adenovirus (Ad-null) Infection (AAV2 group) served as a control. EGFP positive cells were observed by fluorescence microscopy. The percentage of EGFP positive cells and the average fluorescence intensity of single cells were detected by flow cytometry. The expression of EGFP protein was detected by Western blotting to compare the transduction efficiency of each group. The luciferase (Luc) -labeled AAV2 was used to detect the transduction efficiency of the two groups of tumor cells in vitro and the nude mouse tumor model by Luc detection system. Fluorescence quantitative PCR was used to detect the relative copy number of EGFP in DNA and mRNA of infected cells. The effect of Ad-null on AAV2 replication and transcription was observed. Results With the increase of Ad-null titer, the number of EGFP positive cells in AAV2 + Ad-null group was significantly increased. The positive rate of EGFP positive cells counted by flow cytometry was 10.9%, 18.0%, 36.2% and 55.2% at 0.01, 0.1, 1 and 10 MOI, respectively, which was significantly higher than that of AAV2 group (6.4%). Fluorescence intensity of single cells in AAV2 + 10 MOI Ad-null group was 1.32 times of AAV2 group. The protein expression of EGFP in AAV2 + Ad-null group was also higher than that in AAV2 group. Both in vitro and in vivo Luc assays showed that Ad-null significantly enhanced the transduction efficiency of AAV2 on tumors, with 28 and 4.5 folds of fluorescein as compared to AAV2 alone. Ad-null increased the level of AAV2 mRNA in tumor cells. The copy number of EGFP in AAV2 + Ad-null group was significantly higher than that in AAV2 group when Ad-null titer was 1 and 10 MOI, while Ad-null had no effect on AAV2 The effect of DNA was insignificant, and the difference of EGFP copy number between AAV2 + Ad-null group and AAV2 group was not significant. Conclusions Combined use of a small amount of adenovirus can significantly improve the transduction efficiency of AAV on tumor cells, which may be related to the increase of AAV transcription level. It provides experimental evidence for future application of AAV as a carrier for gene therapy of tumors.