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目的构建乏氧/辐射双敏感启动子介导的增强型绿色荧光蛋白表达载体,探讨HRE对A549细胞中Egr-1启动子在乏氧和常氧条件下辐射诱导表达的影响。方法利用化学合成HRE上、下链,PCR扩增得到双链HRE;利用基因重组技术构建HRE/Egr-1双敏感启动子介导增强型绿色荧光蛋白的表达载体pcDNA3.1-HRE/Egr-1-EGFP,经酶切、PCR和测序鉴定正确后,质粒经脂质体介导转染肺腺癌A549细胞,不同剂量照射和不同浓度乏氧后,流式细胞术检测EGFP的表达,以此反映Egr-1的表达特性。结果酶切、PCR和测序鉴定pcDNA3.1-HRE/Egr-1-EGFP质粒构建正确。流式细胞术结果显示常氧条件下,不同剂量X射线照射后,A549细胞中EGFP表达在随着时间延长而逐渐增加,2Gy和4 Gy照射后12 h时达到最大,而6 Gy、8 Gy和10 Gy照射后在8 h时达到最大,而后逐渐降低,与0 h表达比较具有显著性差异(P<0.05,P<0.01)。0.1%-2.5%的氧浓度条件下,EGFP表达随着氧浓度和剂量的增加而逐渐增加,在1%氧浓度时表达量最大,与常氧条件下不同剂量照射后8 h表达比较具有显著性差异(P<0.05,P<0.01);在5%-10%氧浓度条件下,EGFP表达与常氧条件下基本一致。结论 HRE具有增强A549细胞中Egr-1启动子诱导表达的特性,对肿瘤基因-放射治疗具有参考意义。
OBJECTIVE: To construct enhanced green fluorescent protein (EGFP) expression vector mediated by hypoxia / radiation dual sensitive promoter and to explore the effect of HRE on radiation-induced expression of Egr-1 promoter in hypoxic and normoxic A549 cells. Methods The double-stranded HRE was amplified by chemical synthesis of the upper and lower strands of HRE, and the recombinant expression vector pcDNA3.1-HRE / Egr-1 was constructed by using gene recombination technology to construct the double green fluorescent protein (EGFP) 1-EGFP. After identification by restriction enzyme digestion, PCR and sequencing, plasmid was transfected into lung adenocarcinoma A549 cells by lipofectamine. After different doses of radiation and different concentrations of hypoxia, the expression of EGFP was detected by flow cytometry This reflects the expression characteristics of Egr-1. Results The recombinant plasmid pcDNA3.1-HRE / Egr-1-EGFP was identified by restriction enzyme digestion, PCR and sequencing. Flow cytometry showed that under normoxia, the expression of EGFP in A549 cells increased gradually with the increase of time under the different dose of X-ray irradiation, reached the maximum at 12 h after 2Gy and 4 Gy irradiation, while the expression of EGF, And reached the maximum at 8 h after irradiation with 10 Gy, and then gradually decreased. There was a significant difference (P <0.05, P <0.01) compared with that at 0 h. The expression of EGFP increased with the increase of oxygen concentration and dose under the oxygen concentration of 0.1% -2.5%. The expression of EGFP reached its peak at 1% oxygen concentration and was significantly higher at 8 h after exposure to different doses of normoxia (P <0.05, P <0.01). Under the condition of 5% -10% oxygen concentration, the expression of EGFP was basically consistent with that under normoxia. Conclusion HRE has the characteristic of enhancing the expression of Egr-1 promoter in A549 cells, which is of reference value to tumor gene-radiation therapy.