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目的构建双基因表达质粒pEgr-IFNγ-endostatin并检测其在Lewis肺癌细胞中的辐射诱导表达。方法利用基因重组技术构建含Egr-1启动子的IFNγ和endostatin双基因表达质粒,脂质体介导体外转染Lewis肺癌细胞,用酶联免疫吸附法(ELISA)检测不同剂量X射线诱导IFNγ和endostatin表达的量效关系和时程。结果酶切鉴定证实Eg-r1启动子、IFNγ和endostatin基因正确插入双基因表达载体pIRES1neo;不同剂量X射线照射转染该重组质粒的Lewis肺癌细胞上清中IFNγ和endostatin表达量明显高于假照组(均P<0.001),其中5Gy照后表达量最高,分别为假照组的4.14倍和2.92倍;2GyX射线照后Lewis肺癌细胞上清中IFNγ和endostatin含量随时间延长而增加,照后36h分别为照射前的3.75倍和3.02倍(均P<0.001)。结论本实验成功构建了双基因表达
Objective To construct a double gene expression plasmid pEgr-IFNγ-endostatin and to detect its radiation-induced expression in Lewis lung cancer cells. Methods Recombinant plasmids of IFNγ and endostatin containing Egr-1 promoter were constructed by gene recombination technique. Lipofectamine was used to transfect Lewis lung carcinoma cells in vitro. The effects of different doses of X-ray irradiation on IFNγ and IL-6 expression were detected by enzyme-linked immunosorbent assay (ELISA) Efficacy and time course of endostatin expression. Results The restriction endonuclease digestion confirmed that Eg-r1 promoter, IFNγ and endostatin genes were correctly inserted into the double-gene expression vector pIRES1neo. The expression levels of IFNγ and endostatin in Lewis lung cancer cells transfected with the recombinant plasmids at different doses of X-ray irradiation were significantly higher than those of the control (All P <0.001). The highest expression level was found in 5 Gy after irradiation, which were 4.14 and 2.92 times higher than those in sham group respectively. The content of IFNγ and endostatin in supernatant of Lewis lung cancer cells increased with time after 2Gy X-ray irradiation. 36h were 3.75 times and 3.02 times before irradiation (both P <0.001). Conclusion The experiment successfully constructed a double gene expression