肿瘤坏死因子α调控启动子活性促进netrin-1的表达

目的 探讨TNF-α对netrin-1基因表达的影响，并对其调控机制作初步研究。方法 使用RT-PCR和Western blot方法检测TNF-α对netrin-1表达影响。PCR反应克隆出netrin-1启动子序列，构建pGL3-netrin-1-promoter荧光素酶报告基因表达载体。转染HEK-293A细胞检测荧光素酶活性。观察细胞因子TNF-α对netrin-1转录水平的表达调控。结果 首先证实了TNF-α能够上调Netrin-1的表达。其次成功构建pGL3-netrin-1-promoter荧光素酶表达载体，测序正确，然后利用双荧光素酶报告基因系统证实构建的报告基因载体启动子具有活性。发现肿瘤坏死因子TNF-α可以使netrin-1启动子活性显著增加 (P<0.05)，TNF-α以剂量和时间依赖方式调节netrin-1启动子的活性。结论 细胞因子TNF-α可能通过调控启动子的活性而调节netrin-1的表达。

TNF-α regulates netrin-1 expression through enhancing promoter activity

Objective To investigate the regulatory effect of TNF-α on netrin-1 expression and the related mechanism.Methods The expression of netrin-1 was examined by RT-PCR and Western blotting analysis in HEK-293 cells treated with TNF-α.The netrin-1 gene promoter was amplified by PCR and was cloned into pGL3 basic vector.Then the purified pGL3-netrin-1-promoter was transfected into HEK-293 cells,the luciferase activity of netrin-1 gene promoter was detected by luminometer,and the regulatory effect of TNF-α on netrin-1 expression was observed.Results It was confirmed that TNF-α increased expression of netrin-1.pGL3-netrin-1-promoter vector was successfully constructed as confirmed by sequencing.The pGL3-netrin-1-promoter vector was confirmed to have the promoter activity as confirmed by the luciferase system.The activity of netrin-1 promoter was increased significantly in HEK-293 cells after treated with TNF-α(P<0.05),and the increase was in a time-and concentration-dependent manner.Conclusion TNF-α can regulate netrin-1 expression through modulating netrin-1 promoter activity.