pVAX-iNOS真核表达载体的构建及其抗肺癌作用研究

摘要

背景与目的 iNOS与NO介导的抗肿瘤效应有关.本研究旨在构建pVAX-iNOS载体并转染A549肺癌细胞,检测其基因的表达并初步探讨iNOS基因表达增高后对A549肺癌细胞的抗肿瘤作用.方法 应用RT-PCR方法扩增人iNOS编码序列的CDS片段,构建pVAX-iNOS载体后转染肺癌A549细胞,通过RT-PCR和Western blot方法检测目的基因的表达；采用MTT法、Hoechst 3235染色和划痕实验分别检测iNOS高表达在体外对肺癌A549细胞增殖、凋亡和迁移作用的影响.结果 真核表达质粒载体pVAX-iNOS构建成功,iNOS蛋白在转染后的A549细胞中表达升高.pVAX-iNOS转染A549肺癌细胞后能明显诱导细胞发生凋亡并抑制肿瘤细胞的生长和迁移.结论 本研究成功构建pVAX-iNOS真核表达质粒,高表达iNOS能明显抑制A549细胞的增殖、迁移并促进细胞发生凋亡.本研究有望为临床治疗肺癌提供一个新的有效策略.%Background and objective The iNOS gene is associated with NO-mediated antitumor effects. The aims of this study are to construct a eukaryotic expression plasmid that carries the iNOS gene and to detect the expression levels and antitumor effects of the iNOS gene on A549 lung cancer cells. Methods A DNA fragment of the human iNOS coding sequence was amplified using reverse transcription polymerase chain reaction (RT-PCR). The DNA fragment was subsequently cloned into the multiple cloning sites of the eukaryotic expression vector pVAX. The recombinant plasmid was confirmed using restriction enzyme treatment, PCR, and sequencing and was then transfected into A549 lung cancer cells. The expression of the iNOS gene in the AS49 lung cancer cells after transfection was verified by RT-PCR and Western blot analysis. The effects of iNOS on cell apoptosis, proliferation, and migration were identified by staining with Hoechst 3235, an MTT assay, and a scratch assay, respectively. Results The results of the restriction enzyme digestion, PCR, and sequencing verified the successful construction of the eukaryotic expression plasmid pVAX-iNOS. The iNOS gene expression level was increased in the transfected A549 cells. Further experiments also showed increased cell apoptosis among the AS49 lung cancer cells transfected with pVAX-iNOS. Meanwhile, the proliferation and migration of AS49 cells were significantly inhibited by the enhanced iNOS gene expression. Conclusion The recombinant eukaryotic expression vector pVAX-iNOS was successfully constructed and transfected into AS49 cells. The enhanced iNOS gene expression significantly promoted cell apoptosis, whereas the proliferation and migration of A549 cells were inhibited. These findings contribute to the development of novel and effective gene therapies for lung cancer.