论文部分内容阅读
目的构建放射敏感性启动子调控的凋亡素(Apoptin)基因的真核表达质粒pE6-Apoptin-EGFP,探讨其在X线调控下对肺腺癌细胞A549的杀伤作用。方法分别以限制性内切酶EcoRⅠ/NotⅠ双酶切质粒pE6-p53-EGFP及pApoptin-EGFP,电泳分离,回收目的线性DNA片段,T4连接酶连接,构建重组质粒pE6-Apoptin-EGFP,测序、鉴定。脂质体介导瞬时转染肺腺癌细胞A549,RT-PCR、荧光显微镜等检测照射前后融合蛋白表达,流式细胞仪检测放射诱导前后转染细胞的凋亡变化。结果成功构建重组质粒pE6-Apoptin-EGFP,并在被转染A549细胞中检测到Apoptin基因表达;经放射诱导的pE6-Apoptin-EGFP/A549细胞及对照组pE6-EGFP/A549细胞的凋亡率分别为(32.48±3.56)%和(10.67±2.42)%,未经放射诱导的pE6-Apoptin-EGFP/A549细胞及对照pE6-EGFP/A549细胞的凋亡率分别为(6.33±1.21)%、(4.25±0.87)%;与其它3组相比,放射诱导的pE6-Apoptin-EGFP/A549细胞凋亡率明显增高,具有非常显著统计学差异(P<0.01)。结论放射敏感性启动子调控的Apoptin基因表达可在放射线调控下在A549细胞中有效表达并诱导凋亡,为进一步研究非小细胞肺癌的放射-基因联合治疗奠定了基础。
Objective To construct eukaryotic expression plasmid pE6-Apoptin-EGFP of Apoptin gene regulated by radiosensitizer and investigate its cytotoxicity on human lung adenocarcinoma A549 cells under X-ray irradiation. METHODS: Plasmids pE6-p53-EGFP and pApoptin-EGFP were digested with EcoRⅠ / NotⅠ restriction enzymes, respectively. The target linear DNA fragments were isolated by electrophoresis and ligated with T4 ligase to construct recombinant plasmid pE6-Apoptin-EGFP. Identification. The expression of fusion protein was detected by RT-PCR and fluorescence microscopy after transient transfection of lung adenocarcinoma A549 cells. The apoptosis of transfected cells was detected by flow cytometry. Results The recombinant plasmid pE6-Apoptin-EGFP was successfully constructed and the expression of Apoptin gene was detected in the transfected A549 cells. The apoptosis rate of pE6-EGFP / A549 cells induced by irradiation and pE6-EGFP / A549 cells (P <0.05). The apoptosis rates of pE6-Apoptin-EGFP / A549 cells and control pE6-EGFP / A549 cells without radiation were (6.33 ± 1.21)% and (4.25 ± 0.87)%. Compared with the other three groups, the apoptotic rate of pE6-Apoptin-EGFP / A549 cells was significantly increased (P <0.01). Conclusion The expression of Apoptin regulated by radiosensitive promoter can be efficiently expressed and induced apoptosis in A549 cells under the regulation of radiation, which lays the foundation for further study of combined radiotherapy and gene therapy in non-small cell lung cancer.