HRE对A549细胞中Egr-1启动子辐射诱导表达的影响

The effects of HRE on Egr-1 promoter inducing expression by radiation

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 expression vector pcDNA3.1-HRE/Egr-1-EGFP mediated by hypoxia/radiation dual-sensitive promoter,and to approach the effects of HRE on Egr-1 promeoter inducing expression by radiation under hypoxia and normoxia condition.Methods HRE upper and lower strands were gotten by chemical synthesis,double strands HRE were gotten by PCR;HRE/Egr1 double sensitive promoter mediated enhanced green fluorescent protein expression vector pcDNA3.1-HRE/Egr1-EGFP was constructed by gene recombination technique,it was identified correctlly by enzyme digestion,PCR and sequencing.Then these plasmids were transfected into A549 cells by liposome,after different doses X-rays exposure and different oxygen concentration culture,the EGFP expression was measured by cytometry flow,the expression could indicate the Egr-1 expression character.Results pcDNA3.1-HRE/Egr-1-EGFP plasmid was constructed correctly with enzyme digestion,PCR and sequencing.The cytometry flow results showed that EGFP expression in A549 cells increased with time prolongation after different doses X-rays exprosue under normoxia condition,it reached the maximum 12 h after 2 Gy and 4 Gy irradiation,but 8 h after 6 Gy,8 Gy and 10 Gy irradiation,then decreased gradually,it had significant difference comparing with the expression 0 h after irradiation （P〈0.05,P〈0.01）.Under 0.1%-2.5% oxygen,the EGFP expression increased with oxygen concentration and radiation dose increase,it reached maximum under 1% oxygen,it had significant difference comparing with the expression 8 h after different X-rays irradiation under normoxia condition（P〈0.05,P〈0.01）;under 5% -10% oxygen,EGFP expression was similar with normaxia condition.Conclusion HRE has the property to enhance Egr-1 promoter inducing expression by radiation in A549 cells,and has reference significance for tumor gene-radiotherapy.