靶向VEGF基因siRNA对放射性核素杀伤肿瘤的增强作用

目的建立特异性靶向VEGF基因表达的siRNA质粒，在肿瘤细胞内诱导RNA干扰，抑制VEGF表达，观察siRNA对放射性核素杀伤肿瘤的增强作用。方法构建针对人VEGF mRNA干扰质粒载体pRNAH—VEGF和对照载体pRNA—Ctr并转染人乳腺癌的MCF-7细胞株。采用RT—PCR和Western blot检测VEGF在mRNA和蛋白水平的表达抑制作用。用放射性核素^32P胶体作为放射源照射细胞24h，采用克隆形成实验分析siRNA对放射性核素杀伤肿瘤的增强作用。结果与对照组相比较，转染了pRNAH（VEGF的MCF-7细胞VEGF mRNA和蛋白质抑制率均明显下调，细胞培养液上清中VEGF浓度下降了60．6％；siRNA和放射性核素联合使用组细胞克隆数量明显少于各自单独使用组；联合使用组细胞凋亡率也高于单独使用组。结论针对人VEGF mRNA干扰质粒载体有效地抑制内源性VEGF表达，增强了放射性核素对肿瘤细胞的杀伤作用。利用RNA干扰技术抑制VEGF表达可作为肿瘤放射治疗中的辅助治疗，以增加疗效。

Enhanced Radiotherapy-Mediated Killing of Human Cancer Cells by Small Interfering RNA Silencing of Vascular Endothelial Growth Factor

Objective To develop a DNA vector-based RNA interference (RNAi) technology that inhibits the vascular endothelial growth factor (VEGF) expression in order to confer enhanced radiosensitivity to tumour cells.(Methods )mRNA interfering double-stranded DNA vector pRNAH- VEGF and control vector pRNA- Ctr were constructed respectively,and then were transfected into MCF-7 cells. Irradiation was conducted by adding colloid of chromic ~(32)P phosphate (~(32)P-CP) into MCF-7 cells culture system. The expression of VEGF was analyzed RT-PCR and Western blot in MCF-7 cells after transfected by siRNA. Clonogenic assays and apoptosis assays were used to evaluate the effect of irradiation. Results siRNA-VEGF led to a significant reduction in VEGF protein expression in MCF-7 in comparison to controls. ELISA showed that the inhibition rate of pRNAH-VEGF on VEGF in the supernate was 60.6%. While clonogenic assays showed only slight decreases in colony formation in cells treated with siRNA-VEGF alone, the combination with ~(32)P-CP resulted in a significantly enhancement of ~(32)P-CP toxicity in MCF-7 cells, which was associated with the increase of tumour cell apoptosis. Conclusion These data provided a strong evidence for the potential use of siRNA targeting to VEGF as a novel radiotherapy sensitizing agent.