摘要
目的探讨腺病毒介导血管内皮生长因子受体(KDR)启动子驱动的双自杀基因(CDglyTK)联合 survivin 反义寡核苷酸(ASODN)对乳腺癌细胞(MCF-7)及血管内皮细胞(ECV304)的特异性杀伤作用。方法将质粒 pAdEasy-KDR-CDglyTK 在293细胞内包装、扩增后,体外感染表达KDR 的 MCF-7和 ECV304细胞株,同步用 survivinASODN 转染已感染腺病毒的 MCF-7和 ECV304细胞株,观察腺病毒的感染效率和 survivinASODN 的转染情况,应用逆转录聚合酶链反应(RT-PCR)和Western blot 免疫印迹法检测 CDglyTK 和 survivin 基因在转基因 MCF-7和 ECV304细胞中的表达,MTT 法测定两者联合应用对细胞的杀伤效应和旁观者效应。结果 SurvivinASODN 可转染重组腺病毒感染的细胞,并且 survivinASODN 的转染率及重组腺病毒的感染率均不受影响,CDglyTK 可在两种细胞中高表达,survivinASODN 可明显降低 survivin 蛋白表达。单一 survivinASODN 基因转染 MCF-7和 ECV304细胞后,细胞存活率为56.4%±3.8%和55.9%±3.6%,与 AdKDR-CDglyTK 基因联用后,随着丙氧鸟苷(GCV)和5-氟胞嘧啶(5-FC)浓度的增加,细胞存活率迅速下降,两者联合作用与单一基因作用相比,差异有统计学意义(P<0.05)。但在 GCV 100μg/ml、5-FC 2000μg/ml 时,联合基因治疗组细胞存活率略低于单用 AdKDR-CDglyTK 组,两者差异无统计学意义(P>0.05)。SurvivinASODN 和 AdKDR-CDglyTK 联合作用在 GCV、5-FC 浓度较低时表现为协同效应,并具有更明显的旁观者效应。结论 KDR 启动子调控的 CDglyTK 融合基因体系和 survivinASODN 基因联合应用对乳腺癌细胞及血管内皮细胞具有显著的特异性杀伤作用。
Objective To evaluate the selectively killing effects of combination of adenovirus mediated double suicide gene driven by kinase-domain insert containing receptor （KDR） promoter and survivin antisense oligonucleotide on breast tumor cells and vein endothelial cells. Methods Human embryonal kidney cells 293 were transfected with the plasmids of pAdEasy-KDR-CDglyTK and the adenovirus was generated. The KDR expressive cells of MCF-7, ECV304 were infected by adenovirus and survivinASODN was transfered into the same cells meanwhile. The infection rates of adenovirus and transfection efficiency of survivinASODN were observed and the expression of CDglyTK was detected by RTPCR. The expression of survivin was measured by Western blot. The killing effects and bystander effects on cells were assessed by MTY assay. Results The cells infected by the adenovirus mediated double suicide gene could be transfected with the survivinASODN and the infection rate was not affected as well as the transfection rate. The high expression of CDglyTK gene was found in the two kinds of cells and survivinASODN decreased the survivin protein level. When survivinASODN was transfered into MCF-7, ECV304 cells,the survival rates were 56. 4%±3.8% and 55.9% ±3.6% respectively. The combination of survivinASODN and AdKDR-CDglyTK gene therapy showed significantly lower survival rate comparing with using each treatment alone （ P 〈 0. 05 ） and the survival rate decreased gradually with the increasing of the concentration of GCV and 5-FC. But the survival rate for combined gene therapy group was slightly lower in GCV 100 μg/ml, 5-FC 2000 μg/ml than that of AdKDR-CDglyTK group （ P 〉 0. 05 ）. The combination of survivinASODN and AdKDR-CDglyTK therapy showed synergistic killing efficacy and more significant bystander effects. Conclusion The combined therapy with AdKDR-CDglyTK system and survivinASODN shows obvious killing effects on breast tumor cells and vein endothelial cells.
出处
《中华外科杂志》
CAS
CSCD
北大核心
2007年第7期476-479,共4页
Chinese Journal of Surgery
关键词
乳腺肿瘤
血管内皮细胞
基因
反义寡核苷酸
Breast neoplasms
Vascular endothelial
Gene
Oligoribonueleotides,antisense
