Ad-VEGFP-CD/TK系统抑制乳腺癌细胞增殖的体内外实验研究

VEGF-mediated double suicide gene selectively kills breast cancer cells MCF-7

目的探讨腺病毒介导VEGF启动子驱动的CD/TK双自杀基因体系(Ad-VEGFP-CD/TK)对乳腺癌细胞MCF-7的体内外靶向杀伤作用。方法用重组腺病毒Ad-VEGFP-CD/TK体外感染表达VEGF的MCF-7细胞和不表达VEGF的原代培养的乳腺上皮细胞,荧光显微镜观察其感染率,然后给予前药GCV和5-FC,用MTT法观察该体系对细胞生长增殖的影响及其旁观者效应;用流式细胞术观察细胞周期及细胞内DNA含量的变化。建立MCF-7裸鼠皮下移植瘤模型,瘤内注射Ad-VEGFP-CD/TK,腹腔注射前药14d,观察肿瘤生长抑制效应。结果腺病毒对两种细胞的感染率相似。MTT法检测显示MCF-7细胞对前药具有较高的敏感性,而乳腺上皮细胞对前药不敏感,且观察到该体系对MCF-7细胞明显的旁观者效应。在感染复数为100时,用流式细胞仪测定用药组出现典型的凋亡峰;细胞周期分析显示治疗后细胞G0-G1期比率增多,G2-M及S期细胞减少。在MCF-7裸鼠移植瘤模型中,该双自杀基因系统能够显著抑制肿瘤的生长。结论VEGF启动子可调控双自杀基因体系选择性杀伤人乳腺癌细胞MCF-7并诱导细胞凋亡,并可显著抑制人乳腺癌裸鼠移植瘤的生长。

Objective To study the selective killing effect of adenoviru（Ads） - mediated double suicide gene （CD/TK） under the regulation of the human vascular endothelial growth factor （VEGF） promoter on breast cancer cells MCF - 7, Methods The VEGF - expressing MCF - 7 cells and non VEGF - expressing normal human mammary epithelial cells in primary culture were infected by the Ad - VEGFP - CD/TK, The infection efficiencies were observed under a fluorescence microscope. Followed by treatment with GCV and 5 - FC, the killing effects were evaluated and bystander effects were analyzed by MTT assay. Distribution of cell cycle and DNA content in MCF - 7 were detected by flow cytometry. An animal model of human breast cancer in nude mice was reproduced. Ad - VEGFP - CD/TK was injected directly into the tumor. Subsequently, the nude mice were given intraperitoneal injection of GCV[50 mg/（ kg · d） ] and 5 - FC [500 mg/（kg · d） ] daily for 14 days. The inhibition rates for tumor growth were also calculated. Results The infection rates of the resultant recombinant Ads to MCF - 7 and human mammary epithelial cells were not significantly different, and the rates increased gradually with the addition of multiple of infection （MOI） of Ads. MTT assay showed ： MCF - 7 cells infected with Ads were highly sensitive to the pro - drugs, while the infected human mammary epithelial cells were not sensitive. There was considerable bystander effect in MCF - 7 cells. At the multiplicity of infection （MOI） of 100, apoptotic peak was also shown in pro - drug group by flow cytometry. The analysis of cell cycle revealed that the rate of cells at the G0 - G1 phase increased and the rate at the G2 - M and S phase decreased by treatment with pro - drug using flow cytometry. Tumors size decreased during treatment of double suicide gene system by establish subcutaneous transplant model. Conclusion The CD/TK fusion gene system controlled by VEGF promoter has selective killing effects on the VEGF - expressing MCF -7 cells and induces cell apoptosis. It significantly inhibits the growth of xenografted breast cancer in nude mice.