乏氧/辐射双敏感启动子介导分泌型人TRAIL基因对肺腺癌A549细胞凋亡的影响

Effect of secreting type human trail gene on apoptosis of A549 cell lung adenocarcinoma mediated by hypoxia/radiation dual-sensitive promoter

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 secreting type human Trail（shTrail） gene vector pcDNA3.1-HRE/Egr1-shTrail mediated by hypoxia/radiation dual-sensitive promoter,and observe the effect on changes of A549 cell apoptosis and Caspase-3 expression.METHODS：HRE upper and lower chains were got by chemical synthesis,and double strands HRE were got by PCR; HRE/Egr1 dual-sensitive promoter mediated shTrail expression vector pcDNA3.1-HRE-Egr1-shTrail was constructed by gene recombination technique,after it was identified correctlly by enzyme digestion,PCR and sequencing,pcDNA3.1-HRE-Egr1-shTrail was transfected into A549 cells,the cell apoptosis was measured with flow cytometry and TUNEL,Caspase-3 expression was detected with Western blot.RESULTS：The plasmid length was 6 282 bp,after enzyme digestion by BamH Ⅰ and Sma Ⅰ,the fragments that their lengths were 1 284 and 4 998 bp,2 292 and 3 990 bp were got; the vector was amplificated by PCR with Egr1 and shTrail primer,the products that their lengths were 469 and 820 bp; pcDNA3.1-HRE/Egr1-shTrail was sequenced,the result showed the sequence was the same as the design,and this demonstrated the construction was right.After transfection,hypoxia and radiation increased A549 cell apoptotic percentage （P0.05 and P0.01）,induced the Caspase-3 protein expression increase,when both were combined,the effects were more obvious （P0.01）; and hypoxia and radiation increased the Caspase-3 expression （P0.05 and P0.01）,when both were combined,the effects were more obvious （P0.01）.CONCLUSION：Under the condition of hypoxia and radiation,secreting type human Trail（shTrail） gene vector mediated by hypoxia/radiation dual-sensitive promoter can increase A549 cell apoptosis,induce the Caspase-3 expression increase,and the effect of both unification is more obvious.