放射敏感性启动子调控Apoptin基因表达对肺腺癌细胞的杀伤作用研究

Apoptotic effects of Apoptin gene expression regulated by radiosensitive promoter Egr-1 on human pulmonary adenocarcinoma cells

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。

We aimed to construct the eukaryotic expression plasmids of pE6-Apoptin-EGFP containing radiosensitive promoter of Egr-1 and target gene Apoptin to observe its effects on the apoptosis of human pulmonary adenocarcinoma cells line A549.The eukaryotic expression plasmids of pE6-p53-EGFP and pApoptin-EGFP were digested by restriction enzyme EcoR I and Not I,and the Apoptin gene was inserted into the digested pE6-p53-EGFP vector to reconstruct the pE6-Apoptin-EGFP plasmid.After the recombinant plasmid was transfected into A549 cell line,the expression of fusion protein Apoptin-EGFP was detected by RT-PCR and fluorescence microscope.The apoptosis of transfected cells was analyzed by flow cytometry assay.Results showed that the plasmid pE6-Apoptin-EGFP was constructed successfully and the expression of Apoptin gene was detected in A549 cells.The apoptotic rates were 6.33% and 4.25% in pE6-Apoptin-EGFP and pE6-EGFP transfected A549 cells,respectively;however,after irradiation,the apoptotic rates were significantly enhanced to 32.48% and 10.67% in the transfected cells,respectively.Results suggest that the expression of Apoptin gene could be regulated successfully by radiosensitive promoter of Egr-1 in A549 cells and the apoptosis could be induced effectively,which could provide experimental data in the field of non-small lung cancer radiation-gene therapy.