摘要
目的构建和鉴定针对VEGF的发卡样siRNA真核表达载体pU-VEGF-siRNA。方法人工合成一对互补并编码相应短发夹状VEGF-siRNA的寡核苷酸链,将其插入到pS ilencerTM2.1-U6neo载体中,经测序鉴定所构建的重组载体是否正确;采用电转染方法将构建的重组载体导入人恶性黑素瘤细胞系A375和人结(直)肠癌细胞系LOVO细胞中,用G418筛选得到稳定表达VEGF-siRNA的细胞;采用逆转录聚合酶链反应(RT-PCR)、ELISA法分别检测转染细胞VEGFmRNA和蛋白的表达变化。结果经测序证明pU-VEGF-siRNA序列正确;G418筛选得到稳定表达VEGF-siRNA的细胞;转染pU-VEGF-siRNA载体后,A375和LOVO细胞VEGF mRNA和蛋白表达均较对照组明显下降(P<0.01)。结论测序结果表明发卡样的VEGF-siRNA真核表达载体构建成功,转染A375和LOVO细胞后获得稳定表达,并可特异性封闭VEGF的表达,为进一步研究pU-VEGF-siRNA载体在恶性黑素瘤治疗中的作用提供了实验基础。
Objective To constructed short interfering RNA （siRNA） eukaryotic expression vector for VEGF and to transfect into malignant melanoma cells. Methods A VEGF-siRNA targeting human VEGF mRNA common sequence was synthesized and was inserted into BamH I-Hind Ⅲ linearized pSllencer^TM neoU6 2.1 vector. The sequence of pU-VEGF-siRNA plasmid was analyzed by DNA sequencer. The recombinant plasmid was transfected into A375 （human malignant melanoma cell line） and LOVO cells （human colorectal carcinoma cell line） by electroporation, and cells with stable expression of VEGF-siRNA were obtained by G418 selection. Results It was verified that the sequence of constructed recombinant plasmid was correct by DNA sequencing. We obtained cells with stable expressions of VEGF-siRNA. The expressions of VEGF mRNA and protein in A375 and LOVO cells of experimental groups were significantly decreased, compared to that of controls （ P 〈 0.01 ）. Conclusion It indicated that hairpin siRNA eukaryotic expression vector for VEGF would be successfully established, and it might play a specific inhibitory role in two kinds of tumor cell lines. At the same time we obtained cells with the stable expression of VEGF-siRNA. This study laid experimental foundation for further research of the therapy of pU-VEGF-siRNA vector in malignant melanoma.
出处
《中国皮肤性病学杂志》
CAS
北大核心
2006年第3期134-137,共4页
The Chinese Journal of Dermatovenereology
关键词
血管内皮生长因子
发卡样siRNA
恶性黑素瘤
构建
表达
Vascular endothelial growth factor
Hairpin siRNA
Malignant melanoma
Construction
Expression
