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目的:研究化疗药物依托泊苷对腺病毒载体介导的外源基因在肿瘤细胞内表达水平的影响。方法:携带外源基因增强型绿色荧光蛋白(EGFP)的复制缺陷型腺病毒Ad5-CMV-EGFP(MOI为1或10)单独或联合终质量浓度为0.2、2、20、40、80、100和200μg/ml的依托泊苷感染体外培养的肿瘤细胞NCI-H446(人非小细胞肺癌细胞株)、A549(人肺腺癌细胞株)、SMMC-7721(人肝癌细胞株)、SGC7901(人胃癌细胞株)、SKBR-3(人乳腺癌细胞株)和BTT(小鼠膀胱移行上皮癌细胞株)后不同时间,流式细胞仪分析肿瘤细胞EGFP阳性率和平均荧光强度,Western blotting检测EGFP蛋白表达,RT-PCR和实时荧光定量PCR检测肿瘤细胞内EGFP的mRNA表达量和DNA拷贝数。结果:不同剂量的依托泊苷可不同程度地提高Ad5-CMV-EGFP在7种肿瘤细胞内的表达水平,但对EGFP阳性率无明显提高。10MOI的Ad5-CMV-EGFP联合40μg/ml依托泊苷分别感染肿瘤细胞NCI-H446、NCI-H460、A549、SMMC-7721、SGC7901、SKBR-3和BTT24h后,细胞内EGFP的荧光强度分别是单独感染的3.3、3.5、3.1、6.2、7.0、5.4和3.4倍。Ad5-CMV-EGFP联合应用依托泊苷后肿瘤细胞内EGFP蛋白表达增加2~5倍,EGFP mRNA表达量提高,但DNA拷贝数未见明显改变。结论:依托泊苷可提高腺病毒载体介导的外源基因在肿瘤细胞内的表达水平,该作用可能是在转录水平上发挥作用的。
OBJECTIVE: To study the effect of chemotherapeutic etoposide on the expression level of adenovirus-mediated exogenous genes in tumor cells. Methods: The replication-defective adenovirus Ad5-CMV-EGFP (MOI of 1 or 10) carrying exogenous gene enhanced green fluorescent protein (EGFP) alone or in combination with the final concentration of 0.2,2,20,40,80,100 (Human non-small cell lung cancer cell line), A549 (human lung adenocarcinoma cell line), SMMC-7721 (human hepatoma cell line), SGC7901 (human Gastric cancer cell lines), SKBR-3 (human breast cancer cell lines) and BTT (mouse bladder transitional epithelial cancer cell lines) at different times, EGFP positive rate and mean fluorescence intensity of tumor cells were analyzed by flow cytometry. EGFP Protein expression, RT-PCR and real-time fluorescence quantitative PCR were used to detect EGFP mRNA expression and DNA copy number in tumor cells. Results: Etoposide at different doses increased the expression of Ad5-CMV-EGFP in 7 kinds of tumor cells to varying degrees, but no significant increase in the positive rate of EGFP. The fluorescence intensity of intracellular EGFP after 10MOI Ad5-CMV-EGFP combined with 40μg / ml etoposide were respectively infected with NCI-H446, NCI-H460, A549, SMMC-7721, SGC7901, SKBR-3 and BTT24h respectively 3.3, 3.5, 3.1, 6.2, 7.0, 5.4 and 3.4 times the infection. Ad5-CMV-EGFP combined with etoposide increased the expression of EGFP protein in tumor cells by 2 to 5 times and increased the expression of EGFP mRNA, but the DNA copy number did not change significantly. Conclusion: Etoposide can increase the expression level of adenovirus-mediated exogenous genes in tumor cells, which may play a role in the transcriptional level.