转录靶向性KDR启动子调控双自杀基因系统对人大细胞肺癌细胞L9981的杀伤作用

Transcriptional Targeting Killing Effect of Human Giant Cell Lung Cancer Cell Lines with CD+TK/GCV+5-FC Driven by KDR Promotor

背景与目的研究KDR启动子转录调控双自杀基因系统(CDglyTK)对人大细胞肺癌细胞L9981的杀伤作用。方法应用分子生物学技术克隆KDR基因的启动子KDRp,构建KDR启动子调控的双自杀基因(CDglyTK)真核表达质粒pcDNA3-KDRp-CdglyTK。并将其导入L9981和人肝癌细胞HepG2中。给予不同的前药(GCV和/或5-FC)处理后,应用MTT法检测各组细胞的存活率。并应用流式细胞仪检测各组细胞的细胞周期和凋亡。结果成功的克隆KDR启动子和构建pcDNA3-KDRp-CdglyTK质粒。pcDNA3-KDRp-CdglyTK转染后,L9981细胞中均可检测到CD和TKmRNA,而HepG2细胞中则无。联合应用5-FC+GCV处理对转双自杀基因细胞(L9981)的杀伤作用显著高于单独应用5-FC或GCV(P<0.05),且二者显示了良好的药物协同作用。结论KDR基因启动子调控双自杀基因系统可以靶向性杀伤人肺癌细胞。

Background and objective To explore the different killing effect to human giant cell lung cancer cell lines L9981 and L9980 with CD＋TK/GCV＋5-FC driven by KDR promotor. Methods KDRp was cloned and an expression vector containing CD-TK gene under the KDR promotor （pcDNA3-KDRp-CdglyTK） was constructed by molecular biological methods, and then was transfected into human giant cell lung cancer cell line L9981 and L9980, and human hepatic cancer cell line HepG2 .the cytotoxicity to these transgenic cells under treatment with GCV and/or 5-FC were measured by MTT assays. In addition ,the tumor suppression effects of different treatments were investigated in nude mice bearing inoculative lung cancer. Results KDRp was successfully cloned and pcDNA3-KDRp-CdglyTK was constructed .CD and TK gene was expressed in L9981 and L9980 cells after pcDNA3-KDRp-CdglyTK transfection, but not in HepG2 cells. The killing effect of the two produrgs used in combination was much stronger than that of exclusive use of GCV or 5-FC（P〈0.05）, There was no cytotoxic effect on the cells transfected with pcDNA3-KDRp-CDglyTK to prodrugs （GCVand/orS-FC） in KDR protein negative cells（p〉0.05）.. In vivo experiment revealed that the tumorigenesis ofL9981 and NL9980 cell transfected CDglyTK are inhibited with combined intraperitoneal injection GCV and 5-FC in nude mouse. Conclusion Tumor targeted expression of CdglyTK gene under the control of KDR promotor represents a novel strategy for safe and effective gene therapy of cancer and might have wide application in the future.