双自杀基因系统诱导乳腺癌细胞凋亡的体内外实验研究  

Adenovirus mediated double suicide gene inducing cell apoptosis in breast cancer cells

作　　者：孔恒[1] 黄宗海[1] 俞金龙[1] 厉周[1] 李强[1] 

机构地区：[1]南方医科大学附属珠江医院普外科,广东广州510280

出　　处：《中国现代医学杂志》2008年第14期1974-1978,共5页China Journal of Modern Medicine

基　　金：国家863计划项目(2001AA217171);广东省自然科学基金项目(013072)

摘　　要：目的探讨VEGF启动子驱动的双自杀基因体系对人乳腺癌细胞MCF-7靶向治疗作用以及该体系能否诱导体内外MCF-7细胞的凋亡。方法选择表达VEGF的MCF-7细胞和不表达VEGF的原代培养的乳腺上皮细胞,用重组腺病毒Ad-VEGFP-CD/TK感染之,荧光显微镜观察其感染效率。RT-PCR方法检测受感染细胞目的基因的表达,给予前药GCV和5-FC后于倒置显微镜观察,采用流式细胞仪检测细胞内DNA含量的变化。建立荷人乳腺癌的裸鼠动物模型,观察各治疗组的治疗效果,应用透射电镜观察移植瘤细胞超微结构。结果携带双自杀基因和报告基因(GFP)的重组腺病毒载体,感染复数为100时,95%以上的受感染MCF-7和乳腺上皮细胞中有GFP表达。RT-PCR检测发现MCF-7细胞有目的基因的表达,而乳腺上皮细胞无表达。倒置显微镜下观察用药组细胞出现凋亡征象;用流式细胞仪测定用药组出现典型的凋亡峰。在MCF-7裸鼠移植瘤模型中,治疗组裸鼠移植瘤的生长明显受到抑制,其余各组肿瘤生长情况无明显差别;在透射电镜下可见治疗组多量的凋亡细胞。结论体内外实验均表明VEGF启动子可调控双自杀基因体系靶向性诱导人乳腺癌细胞MCF-7细胞凋亡。[Objective] To study the selective killing effect of adenovirus （Ad）-mediated fusion gene system driven by human vascular endothelial growth factor （VEGF） promoter on human breast cancer MCF-7 cells and observe whether apoptosis can be induced. [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-CDfI＇K.The infection effi- ciencies were observed under a fluorescence microscope. The expression of CDfrK gene was detected by RT-PCR. The toxic effect induced by giving the prodrug 5-fluorecytosine （5-FC） and ganciclovic（GCV） were determined by light microscopy and flow cytometry （FCM）. An animal model of human breast cancer in nude mice was reproduced. The treatment effect was observed in each group. Then pathological character of tumor growth cells were observed by electronmicroscopy. [Results] The green fluorescent protein（GFP） were observed in the 95 percent of the infected MCF-7 and human mammary epithelial cells when the multiple of infection （MOI） of the Ads are 100. RT-PCR demonstrated that there existed the expression of CDfrK gene in MCF-7 cells infected by Ad-VEGFP-CDTK, while the infected human mammary epithelial cells were not. The progrug could induce apeptosis of MCF-7/CDTK under inverted microscope. Apeptotic peak was also shown in pro-drugs group by flow cytometry. In vivo, tumor growth was dramatically inhibited in treated group. It was no significant difference among the other groups. Morphologic features of xenografts cell apoptosis in the group treated with predrug were displayed via electron microscopy. [Conclusions] The CD/TK fusion gene system controlled by VEGF promoter have selectively killing effect on MCF- 7 cells and inducing the cell apoptesis in vitro and vivo.

关 键 词：自杀基因治疗 转录启动子 细胞凋亡 乳腺癌 腺病毒 

分 类 号：R737.9[医药卫生—肿瘤]