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目的:构建乏氧(HRE)/辐射(Egr1)双敏感启动子介导的分泌型人TRAIL(shTRAIL)基因表达载体pcD-NA3.1-HRE-Egr1-shTRAIL,观察其对肺腺癌A549细胞周期及增殖的影响。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含有HRE/Egr1双敏感启动子介导shTRAIL的表达载体pcDNA3.1-HRE-Egr1-shTRAIL,经酶切、PCR和测序鉴定正确后,干扰质粒经脂质体介导转染肺腺癌A549细胞,利用流式细胞术和TUNEL检测细胞凋亡变化;利用蛋白质印迹法检测Caspase-3蛋白表达。结果:质粒大小为6282bp,BamHⅠ和SmaⅠ酶切,所得片段大小分别为1284和4998、2292和3990bp;以载体为模板,Egr1和shTRAIL引物进行PCR扩增,可得到469bp和820bp产物;将pcDNA3.1-HRE-Egr1-shTRAIL进行测序,所得结果与设计一致,说明构建正确。转染肺腺癌A549细胞后,乏氧与辐射能够增加细胞凋亡的百分比(P<0.05或P<0.01),两者联合,作用更明显,P<0.01;而且乏氧与辐射也能诱导Caspase-3表达的增强(P<0.05或P<0.01),两者联合作用,表达增加更明显,P<0.01。结论:乏氧/辐射双敏感启动子介导分泌型人TRAIL基因在乏氧和辐射条件下能够增加肺腺癌A549细胞的凋亡,诱导Caspase-3蛋白表达的增加,两者联合作用效果更明显。
OBJECTIVE: To construct a recombinant human TRAIL (shTRAIL) gene expression vector pcD-NA3.1-HRE-Egr1-shTRAIL mediated by an HRE / Egr1 double-responsive promoter and observe its effect on lung adenocarcinoma A549 cells Cycle and proliferation effects. Methods: The double-stranded HRE was amplified by PCR using the synthetic HRE. The recombinant plasmid pcDNA3.1-HRE-Egr1-shTRAIL containing the HRE / Egr1 double-responsive promoter was constructed by gene recombination. After PCR and sequencing were identified correctly, the interference plasmids were transfected into lung adenocarcinoma A549 cells by lipofectamine. The changes of apoptosis were detected by flow cytometry and TUNEL. The protein expression of Caspase-3 was detected by Western blot. Results: The size of the plasmid was 6282bp, digested with BamHⅠand SmaⅠ, and the fragments were 1284 and 4998, 2292 and 3990bp in size respectively. The products of 469bp and 820bp were obtained by PCR amplification with Egr1 and shTRAIL primers. 1-HRE-Egr1-shTRAIL, and the results are consistent with the design, indicating that the construction is correct. Transfection of lung adenocarcinoma A549 cells, hypoxia and radiation can increase the percentage of apoptosis (P <0.05 or P <0.01), the combination of the two, the role was more obvious, P <0.01; and hypoxia and radiation can also be induced Caspase-3 expression increased (P <0.05 or P <0.01), the two combined effect, the expression increased more significantly, P <0.01. CONCLUSION: The hypoxia / radiation dual-sensitive promoter can induce the apoptosis of human lung adenocarcinoma A549 cells and induce the increase of Caspase-3 protein expression under the condition of hypoxia and radiation. The combined effect of the two obvious.