联合应用Survivin和VEGF反义寡核苷酸对肺癌A549细胞的抑制作用

Effect of antisense oligonucleotide targeting Survivin and VEGF on the inhibition of A549 cells

目的研究非小细胞肺癌（non-small cell lung cancer,NSCLC）A549细胞经Survivin和VEGF反义寡核苷酸（ASODN）单独和联合转染后细胞生长、凋亡以及对基因表达的抑制作用。方法针对Survivin和VEGF mRNA序列设计反义寡核苷酸,以脂质体（Lip）为载体,介导Survivin和VEGF ASODN单独和联合转染A549细胞。MTT法检测细胞生长抑制率,RT-PCR检测细胞中Survivin和VEGF基因的mRNA含量,流式细胞术检测细胞凋亡以及细胞中Survivin基因的蛋白表达。结果经脂质体转染后200-600 nmol/L Survivin ASODN和5~20μmol/L VEGF ASODN均能抑制A549细胞增殖,诱导细胞凋亡;在48 h时,400 nmol/L Survivin ASODN和10μmol/L VEGF ASODN抑制率和凋亡率有统计学意义（P〈0.05）;两者联合转染A549细胞抑制率和凋亡率分别为（61.37±1.94）%和57.03%,显著高于两基因单独转染（P〈0.05）;联合组的Survivin mRNA的表达量为（15.26±1.31）%,蛋白表达率为（30.40±1.33）%,VEGF mRNA的表达量为（13.12±1.45）%,均显著低于单独转染组（P〈0.05）。结论 Survivin和VEGF ASODN均能抑制A549细胞基因表达,诱导细胞凋亡,两基因反义寡核苷酸联合转染的效果优于单独转染。

[Objective] To investigate cell growth, apoptosis and inhibition of gene expression after trans- fection Survivin antisense oligonueleotides （ASODN） alone or combined with VEGF ASODN on A549 cells. [Methods] We designed a human Survivin and VEGF antisense oligonueleotides. Survivin ASODN alone or combined with VEGF ASODN were transfected into A549 cells by Lipofectin. The MTT assay was used to detect the cell proliferation, mRNA expression of Survivin and VEGF were detected by reverse transcription- polymerase chain reaction （RT-PCR）, the apoptotie rates and protein expression levels of the two genes were analyzed by flow eytometry （FCM）. [Results] After transfected with Lipofectamine, 200- 600 nmol/L Survivin ASODN or 5 - 20 μmol/L VEGF ASODN could inhibited A549 cell proliferation and induced apoptosis; inhibition rate and apoptosis rate was significantly rise at 48 h after A549 cells transfected with 400 nmol/L Survivin ASODN or 10 μmol/L VEGF ASODN （P〈0.05）; after transfected with 400 nmol/L Survivin and 10 μmol/ L VEGF ASODN, the inhibition rate was （61.37 ± 1.94）% and the apoptosis rate was 57.03%, significantly higher than the two genes transfected alone （P〈0.05）; in the combined group Survivin mRNA and protein expression levels was （15.26 ±1.31）% and （30.40 ±1.33）%, VEGF mRNA expression was （13.12 ±1.45）%, which were obviously decreased than transfected alone （P 〈0.05）. [Conclusion] Both Survivin and VEGF ASODN could inhibit the gene expression of A549 cells and induce the apoptosis. The efficiency of co-transfect was better than transfect alone.