Survivin与VEGF基因反义寡核苷酸联合应用人肺腺癌A549细胞作用研究

Effects of antisense VEGF and Survivin oligodexynucleotides on human lung adenocarcinoma cell line A549

摘要:

目的：探讨脂质体介导的血管内皮细胞生长因子（vascular endothelial growth factor，VEGF）与生存素（Survivin）基因反义寡核苷酸联合应用对人肺腺癌细胞系A549生长的影响。方法：构建 VEGF 与 Survivin基因反义寡核苷酸，以脂质体转染人肺腺癌A549细胞。实验分为对照组、脂质体对照组、VEGF反义转染组（VEGF ASODN组）、Survivin反义转染组（Survivin ASODN组）、联合转染组（VEGF ASODN+Survivin ASODN组）。采用RT-PCR检测VEGF与Survivin基因表达；流式细胞仪分析细胞周期和凋亡、检测VEGF与Survivin蛋白表达；MTT观测细胞生长；平板集落实验分析细胞增殖克隆能力。结果：脂质体介导的VEGF与Survivin基因反义寡核苷酸联合转染组A549细胞存活率和增殖克隆能力明显低于其余组，差异有统计学意义（P=0.043，P=0.049，P=0.038，P=0.023）。流式细胞仪分析显示联合转染组细胞周期被阻滞，Survivin蛋白表达低于其余组，与对照组差异有统计学意义（P=0.017，P=0.002），VEGF蛋白表达低于其余组，与对照组差异有统计学意义（P=0.013，P=0.003）。RT-PCR检测结果显示联合转染组VEGF与Survivin基因表达低于其余组。结论：VEGF与Survivin基因反义寡核苷酸联合应用抑制人肺腺癌A549细胞的增殖，延长细胞周期，抑制 VEGF与Survivin基因表达。为后续的动物实验打下了理论依据。

Abstract:

Objective：To investigate the effect of liposome-mediated vascular endothelial growth factor（VEGF） and Survivin antisense oligonucleotide on the growth of human lung adenocarcinoma cell line A549. Methods:VEGF and Survivin antisense oligonucleotides were constructed and human lung adenocarcinoma A549 cells were transfected with LipofectamineTM2000. The experiment was divided into control group，liposome control group，VEGF antisense transfection group（VEGF ASODN group），Survivin antisense transfection group（Survivin ASODN group），joint transfection group（VEGF ASODN+Survivin ASODN group）. VEGF and Survivin were detected by RT-PCR. Flow cytometry（FCM） was applied to measure cell cycle，cell apoptosis and VEGF and Survivin protein. MTT method was applied to measure cell growth. Colony forming test was applied to measure the cell proliferation and clonogenic abilities. Results：A549 cell viability and proliferation and clonogenic abilities were significantly lower in VEGF ASODN+Survivin ASODN group than in the other groups，with significantly statistical differences（P=0.043，P=0.049，P=0.038，P=0.023）. FCM analysis showed that co-trans－fection of transfected cell cycle was arrested and Survivin protein expression was lower in VEGF ASODN+Survivin ASODN group than in the other groups，with significantly statistical differences（P=0.017，P=0.002）. VEGF protein expression was lower in VEGF ASODN+Survivin ASODN group than in the other groups，with significantly statistical differences（P=0.013，P=0.003）. RT-PCR re－sults showed that VEGF and Survivin gene expressions were lower in VEGF ASODN+Survivin ASODN group than in the other groups. Conclusions：Combination of VEGF and Survivn antisense oligonucleotides can inhibit the proliferation of human lung adeno－carcinoma cell line A549，extent cell cycle and inhibit VEGF and Survivin gene expression，which will lay theoretical basis for the subsequent animal experiments.