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目的观察HRE/Egr-1调控的shTRAIL基因对肺腺癌A549细胞的辐射增敏效应。方法将前期构建的含有HRE/Egr-1双敏感启动子介导的shTRAIL基因的质粒pcDNA3.1-HRE/Egr1-shTRAIL经脂质体介导,转染肺腺癌A549细胞,将转染细胞分为3组:乏氧组、照射组和乏氧+照射组,分别进行相应处理,并设正常细胞作为对照。RT-PCR检测各组细胞shTRAIL基因mRNA的转录水平;双抗体夹心ABC-ELISA检测各组细胞上清中shTRAIL的含量;平板克隆实验检测各组细胞的放射敏感性。结果乏氧组、照射组和乏氧+照射组A549细胞shTRAIL基因mRNA的转录水平及细胞上清中shTRAIL蛋白的含量均较正常对照组明显增加,其中,乏氧+照射组最高。常氧条件下,转染质粒pcDNA3.1-HRE/Egr1-shTRAIL的A549细胞和正常对照组细胞的D0值分别为1.89和3.26;而在乏氧条件下,两组的D0值分别为1.13和5.81,表明乏氧条件下质粒转染组A549细胞的放射敏感性最高。结论HRE/Egr-1调控的shTRAIL基因在乏氧和辐射条件下,能够诱导A549细胞中shTRAIL基因mRNA转录和蛋白表达水平增加,并增加细胞的放射敏感性。
Objective To observe the sensitizing effect of shTRAIL gene regulated by HRE / Egr-1 to lung adenocarcinoma A549 cells. Methods The plasmid pcDNA3.1-HRE / Egr1-shTRAIL containing the pre-constructed hTR / Egr-1 double-responsive promoter-mediated shTRAIL gene was transfected into lung adenocarcinoma A549 cells by lipofectamine. The transfected cells Divided into three groups: hypoxia group, irradiation group and hypoxia + irradiation group, respectively, the corresponding treatment, and normal cells as a control. The transcription level of shTRAIL mRNA in each group was detected by RT-PCR. The shTRAIL content in supernatant of each group was detected by double antibody sandwich ABC-ELISA. The radiosensitivity of cells in each group was detected by plate clone assay. Results The transcription level of shTRAIL gene and the content of shTRAIL protein in A549 cells in hypoxia group, irradiation group and hypoxia + irradiation group were significantly higher than those in normal control group, of which hypoxia + irradiation group was the highest. Under normoxic conditions, the D0 values of A549 cells transfected with pcDNA3.1-HRE / Egr1-shTRAIL and normal control cells were 1.89 and 3.26, respectively. Under hypoxic conditions, the D0 values of the two groups were 1.13 and 5.81, indicating that under hypoxia plasmid A549 cells transfected with the highest radiosensitivity. Conclusion Under the condition of hypoxia and radiation, shTRAIL gene regulated by HRE / Egr-1 can induce transcription and protein expression of shTRAIL gene in A549 cells and increase radiosensitivity.