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200 μg/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。10 MOI的Ad5-CMV-EGFP联合40 μg/ml依托泊苷分别感染肿瘤细胞NCI-H446、NCI-H460、A549、SMMC-7721、SGC7901、SKBR-3和BTT 24 h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。

To investigate the effects of chemotherapeutic agent Etoposide on transgene expression mediated by recombinant replication-defective adenovirus in tumor cell lines. Methods：Cultured tumor cells, including NCI-H446, NCI-H460, A549, SMMC-7721, SGC7901, SKBR-3, and BTT were infected by Ad-CMV-EGFP alone （MOI being 1 and 10）or in combination with Etoposide at different final concentrations (0.2, 2, 20, 40, 80, 100 and 200 μg/ml). GFP positive cell rates and the mean intensities of GFP fluorescence in tumor cells were detected by Fluorescence Activated Cell Sorting (FACS) after cultured with different strategies. EGFP protein expression in tumor cells was analyzed by Western blotting. Quantitative analysis of mRNA and DNA copies of EGFP in tumor cells were performed by RT-PCR and real-time PCR. Results: FACS results indicated that Etoposide efficiently enhanced the mean intensities of EGFP fluorescence to different degrees in all 7 cell lines but had no evident effects on the EGFP positive rate. Twenty-four hours after cultured at the presence of 10 MOI Ad5-CMV-EGFP and 40 μg/ml Etoposide, the mean intensities of EGFP fluorescence in NCI-H446, NCI-H460, A549, SMMC-7721, SGC7901, SKBR-3, and BTT cells were respectively 3.3, 3.5, 3.1, 6.2, 70, 5.4, and 3.4 folds that cultured with 10 MOI Ad5-CMV-EGFP alone. Western blotting showed that EGFP protein expression in cells co cultured with Ad5-CMV-EGFP and Etoposide was 2-5 times that cultured with 10 MOI Ad5-CMV-EGFP alone. EGFP mRNA expression had a similar tendency as EGFP protein, but the copies of EGFP DNA had no evident changes. Conclusion: Etoposide can enhance transgene expression mediated by recombination replication defective adenovirus in several tumor cell lines, which may play a role at the transcriptional level.

R730.3

国家自然科学基金杰出青年资助项目（No.30325043）；国家重点基础研究发展计划(973)项目（No.2004CB518804）