医学分子生物学杂志2011，Vol.8Issue(6)：494-500,7.DOI:10.3870/j.issn.1672-8009.2011.06.006
靶向Ang2的RNAi慢病毒载体的构建及其对恶性黑色素瘤细胞中Ang2基因表达的影响
Construction of Recombinant Lentiviral Vector of siRNA for Ang2 and the Effect of the Expression of Ang2 Gene in Malignant Melanoma Cells
摘要
Abstract
Objective To construct lentivectors carrying Ang2-small interfering RNA ( siR-NA ), which was used to inhibit Ang2 expression in malignant melanoma cells. Methods Plasmid pNL-EGFP and recombinant plasmid pSilencer 1. 0-U6-Ang2-siRNA were digested with Xbal and li-gated into a target lentiviral transfer plasmid of pNL-EGFP-U6-Ang2- I or pNL-EGFP-U6-Ang2- Ⅱ . Plasmids pNL-EGFP-U6-Ang2- I and pNL-EGFP-U6-Ang2- II were constructed successfully. Lentiviral vector systems of three plasmids were constituted using pNL-EGFP-U6-Ang2- I ( pNL-EGFP-U6-Ang2- Ⅱ ), pVSVG and pHelper. The lentiviral vector system was transfected into 293T cell to produce pNL-EGFP-U6-Ang2- I and pNL-EGFP-U6-Ang2- Ⅱ lentivirus. Supernatant was collected to determine the titer. Malignant melanoma cells were transduced with both lentiviruses. Realtime RT-PCR was conducted to examine inhibitory efficiency. Results The recombinant lentiviral vectors of Ang2-RNAi were constructed successfully, as confirmed by restriction enzyme digestion and sequencing. Lentiviral titer was up to 8. 0 x l03/ml. Real-time RT-PCR demonstrated that the lentiviral vectors of Ang2-RNAi infect malignant melanoma cells and inhibit the expression of Ang2 genes in malignant melanoma cells ( P < 0. 05 ) . Conclusion Lentivector carrying Ang2-siRNA was constructed successfully and it was able to inhibit the expression of Ang2 gene in vitro significantly. The study established preliminary research on the inhibition of the growth of malignant melanoma xenografts in nude mice and provides the experimental evidence for tumor gene therapy in future.关键词
RNA干扰/Ang2/慢病毒载体/恶性黑色素瘤Key words
RNA interference/ Ang2/ lentiviral vector/ malignant melanoma cells分类
医药卫生引用本文复制引用
刘照亮,王彪,郭国祥,单秀英,王美水,庄福连,蔡传书,张明凤,张彦定..靶向Ang2的RNAi慢病毒载体的构建及其对恶性黑色素瘤细胞中Ang2基因表达的影响[J].医学分子生物学杂志,2011,8(6):494-500,7.基金项目
卫生部科学研究基金-福建省卫生教育联合攻关计划(No.WKJ2008-2-51) （No.WKJ2008-2-51）
