乏氧/辐射双敏感启动子-Smac质粒联合X射线对乏氧A549细胞增殖的影响

Effects of hypoxia/radiation dual-sensitive promoter-Smac plasmid combined with X-rays irradiation on proliferation of hypoxic A549 cells

目的 构建乏氧/辐射双敏感启动子（HRE/Egrl）介导Smac的表达载体,观察其在人肺腺癌A549细胞中常氧和乏氧条件下的表达特点和在乏氧条件下的增殖抑制作用.方法 成功构建表达质粒pcDNA3.l-Egrl-Smac和pcDNA3.1-HRE/Egrl-Smac后转染A549细胞,分别采用实时定量聚合酶链反应（Real-time PCR）和Western blot检测Smac mRNA和蛋白表达;应用噻唑蓝（MTT）实验检测细胞的增殖变化.结果 常氧条件下,与对照组比较,2 Gy照射和转染质粒后进行2 Gy照射能显著增加A549细胞中Smac mRNA表达（P＜0.05）,且乏氧能显著增加这种效应（P＜0.05）;同时,Smac蛋白表达规律与mRNA表达规律相似.常氧和乏氧条件下,2 Gy照射和转染质粒后进行2 Gy照射细胞均较相同时间点对照组细胞增殖水平显著降低（P＜0.05）;而且乏氧各组较常氧各组细胞增殖水平显著降低（P＜0.05）.结论 乏氧和辐射均能诱导A549细胞中Smac表达增加,且联合2 Gy照射能显著抑制乏氧条件下A549细胞的增殖能力.

Objective To construct recombinant expression vector of Smac gene mediated by hypoxia/radiation dual-sensitive promoter （HRE/Egrl）,and observe the expression characteristics in A549 cells under normoxic and hypoxic conditions after transfection,and the inhibitory effects on proliferation under hypoxic condition.Methods The plasmid pcDNA3.1-Egrl-Smac and pcDNA3.1-HRE/Egrl-Smac were successfully constructed,and transfected into A549 cells.The expression levels of mRNA and protein were detected by using real-time quantitative polymerase chain reaction （Real-time PCR） and Western blotting,respectively.Cell proliferation changes were measured by methyl thiazol tetrazolium （MTT） as-say.Results Under normoxic condition,as compared with control group,Smac mRNA was significantly increased in A549 cells irradiated with 2 Gy or transfected and irradiated with 2 Gy （P ＜ 0.05）.Hypoxia could significantly enhance the effects （P ＜ 0.05）.At the same time,Smac protein had similar expression characteristics.Under normoxic and hypoxic conditions,at the same time point,as compared with control group,proliferation was significantly decreased in A549 cells irradiated with 2 Gy or transfected and irradiated with 2 Gy （P ＜ 0.05）,and proliferation in each group under hypoxic condition was more lower than that in each group under normoxic condition （P ＜ 0.05）.Conclusion Both hypoxia and radiation could enhance the Smac expression in A549 cells,and combination of hypoxia and 2 Gy irradiation could significantly inhibit proliferation of A549 cells.