VEGF介导的双自杀基因系统对乳腺癌细胞的靶向杀伤作用

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效率，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GCV(50mg/kg•d)和5-FC(500mg/kg•d)14天，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，其感染率随腺病毒滴度的增高而递增。MTT法检测显示表达VEGF的MCF-7细胞对前药具有较高的敏感性，而不表达VEGF的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。

Adenovirus mediated double suicide gene under controlling by VEGF promoter kills selectively breast cancer cells MCF-7

【Abstract】Objective To study the selectively killing effect of adenovirus (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 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. Then, the nude mice were given intraperitoneal injection of GCV(50mg/kg•d) and 5-FC(500mg/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 apparently 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. And there was considerable bystander effect in MCF-7 cells. At the multiplicity of infection (MOI) of 100, apoptotic peak was also shown in pro-drugs group by flow cytometry. The analysis of cell cycle revealed that the rate of cells at the G0-G1 phase is increasing and the rate at the G2-M and S phase is decreasing by treatment with pro-drug by flow cytometry. Further, we also observed that tumors decreased in size during treatment of double suicide gene system by establish subcutaneous transplanted model. Conclusions The CD/TK fusion gene system controlled by VEGF promoter have selectively killing effect on the VEGF-expressing MCF-7 cells and inducing the cell apoptosis. And it could significantly inhibit the growth xenografted breast cancer of in nude mice.