双自杀基因系统对乳腺癌细胞体外特异性杀伤作用  

Specific Killing Effects of Double Suicide Gene Cytosine Deaminase and Thymidine Kinase on Breast Cancer Cells

作　　者：黄宗海[1] 孔恒[1] 俞金龙[1] 厉周[1] 宋慧娟[1] 杜江[2] 车小燕[3] 

机构地区：[1]南方医科大学附属珠江医院普外科,广州市510280 [2]广州市珠江医院儿科实验室 [3]广州市珠江医院中心实验室

出　　处：《中国肿瘤临床》2008年第2期104-108,共5页Chinese Journal of Clinical Oncology

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

摘　　要：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,并以RT-PCR、Westernblot方法检测受感染细胞CD/TK的表达,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;电镜观察细胞的病变;用流式细胞术观察细胞内DNA含量的变化;分光光度法检测细胞内Caspase-3活性。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。RT-PCR和Westernblot检测发现转染Ad-VEGFP-CD/TK的MCF-7细胞有目的基因和蛋白的表达,而乳腺上皮细胞无表达。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。在感染复数为100时,电镜下可见用药组MCF-7细胞有凋亡改变。用流式细胞仪测定用药组出现典型的凋亡峰;且随着前药浓度的升高转基因细胞内Caspase-3活性逐渐升高。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,细胞内Caspase-3活性升高可能是其诱导细胞凋亡的机制之一。Objective： To investigate the selective killing effects of adenovirus （Ads） mediated double suicide gene （CD/TK） stimulated by vascular endothelial growth factor（VEGF） promoter on breast cancer cells MCF-7. Methods： MCF- 7 cells with VEGF expression and normal human mammary epithelial cells without VEGF expression in primary culture were infected by the Ad-VEGFP-CD/TK. The infection efficiency were observed by a fluorescence microscope. The expression of CD/TK was detected by RT-PCR and Western blot. After treatment with GCV and 5-FC, MTF was employed to evaluate the killing effects. Then pathological features were observed by electron microscope and DNA content in MCF- 7 cells was detected by flow cytometry. The caspase-3 activity was detected by absorption speetrumetry,. Results： The intectiun 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 multiplicity of inffection（MOI） of Ads. RT-PCR and Western blot demonstrated that the product of CD/TK gene existed in MCF-7 cells infected by Ad-VEGFP-CDTK, but not in infected human mammary, epithelial cells. MTT assay showed that MCF-7 cells infected with Ads were highly sensitive to the prodrugs, while the infected human mammary epithelial cells were not. At the MOI of 100, morphologic features of apoptosis in MCF-7 cells were observed by electron microscope. The prodrug administered group showed an apoptotic peak in flow eytometry. Furthermore. the activity of caspase-3 gradually increased with the increasing concentration of the pro-drugs. Conclusion： The CD/TK fusion gene system controlled by VEGF promoter has selective killing effects on MCF-7 cells with VEGF expression and induces apoptosis. The increased activity of easpase-3 may be one of the mechanisms forMCF-7 cell apoptosis induced by pro-drugs.

关 键 词：乳腺癌 基因治疗 腺病毒 凋亡 

分 类 号：R737.9[医药卫生—肿瘤]