重组质粒pcEgr-hp53的构建及在A549和SKOV-3细胞中电离辐射诱导p53基因和蛋白的表达

目的构建重组质粒pcEgr-hp63，并探讨其在A549和SKOV-3细胞中的辐射诱导表达及抗癌作用。方法将野生型p53 cDNA全长插入pcDNA3．1质粒多克隆位点，用Egr-1启动子替代pcDNA3.1质粒CMV启动子，构建成pcEg-hp53重组质粒；通过脂质体介导转染法将重组质粒坶hp53和pcDNA3.1分别转染入A549和SKOV3细胞株，并经G418筛选稳定克隆并扩增培养。采用RT-PCR和流式细胞术的方法检测了不同剂量X-射线照射转染后的A549和SKOV-3细胞053基因转录水平和蛋白表达的变化。结果经限制性内切酶酶切鉴定，辐射诱导表达载体pcEg-hp63构建正确。与0Gy比较，A549-hp53和SKOV-3-hp53经0．5-5Gy照射后，p53mRNA水平增高；A549．hp53在2-5Gy后，p53蛋白表达显著增高（P〈0．01），SKOV-3-hp63其蛋白表达随剂量（0．5-5Gy）增加而明显增加（P〈0．05-P〈0．01）。A549-hp53与A549-vect相同照射剂量组比较，其p53蛋白表达在0．5Gy照射后明显下降（P〈0．05），2-5Gy照射后明显增高（P〈0．01）；SKOV-3-hp53与SKOV-3-vect相同照射剂量组比较，其蛋白表达在0Gy时增高，0.5-5Gy照射后明显增高（P〈0．01）。结论X-射线可激活早期反应生长因子Egr-1，无论肿瘤细胞p53基因状态如何，均可诱导其下游基因p53 mRNA及其p53蛋白表达增强。

Construction of pcEgr-p53 plasmid and its expression induced by radiation in A549 and SKOV-3cells

Objective To construct a recombinant plasmid pcEgr-p53,the expressions of human wild type p53 （hwt-p53） mRNA and protein induced by irradiation in A549 and SKOV-3 dells and its effect in cancer suppression were investigated.Methods hwt- p53 gene was inserted into pcDNA3.1. CMV promoter of pcDNA3.1 was repheed by Egr-1 promoter to construct an expression vector poEgr-hp53, pcEgr-hp53 and pcDNA3.1 were transfected into A549 and SKOV3 cells in vitro. The transfected cells were selected through CA18.The expressions of p53 mRNA and protein were used to detect with RT-PCR and flow eytometry after different doses of X-ray irradiation.Results pcEgr-hp53 expression vector has been constructed comectly. The level of p53 mRNA in AS49-hp53 和 SKOV-3-hp53 increased after irradiation with 0.5-5 Gy.The expressions of p53 protein A549-hp53 with 2- 5 Gy （P 〈0.01） and for SKOV-3-hp53 with 0.5 - 5 Gy （ P 〈 0.05 - P 〈 0.01） were significantly higher than those with 0 Gy for. The expression of p53 protein in A549-hp53 with 0.5 Gy was markedly lower than that in A549-vect with corresponding irradiation doses （ P 〈 0.01）, while with 2- 5 Gy were markedly higher （ P 〈 0.01）. The expression of p53 protein in SKOV-3-hp53 with 0.5 - 5 Gy were markedly higher than that in SKOV-3-vect with corresponding irradiation doses （ P 〈 0.01）. Conclusion The results suggest that X-ray imadiaton could activate Egr-1 gene promoter and induce the expression of downstream gene p53 whether p53 gene in tumor ceils is normal or abnormal.