| 摘要: |
| 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 采用RT-PCR和蛋白质免疫印迹法检测TNF-α对netrin-1表达的影响。用PCR反应扩增netrin-1启动子序列,克隆至pGL3荧光素酶报告基因载体，转染HEK-293A细胞，通过检测荧光素酶活性观察TNF-α对netrin-1转录水平的表达调控。结果 TNF-α(20 ng/ml)能上调netrin-1的表达。成功构建pGL3-netrin-1-promoter荧光素酶表达载体,并证实构建的报告基因载体启动子具有活性；发现TNF-α可使netrin-1启动子活性增加(P<0.05),并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。 |
| 关键词: netrin-1 肿瘤坏死因子α 转录 启动子 |
| DOI：10.3724/SP.J.1008.2012.00613 |
| 投稿时间：2012-02-05修订日期：2012-05-09 |
| 基金项目:辽宁省科技攻关课题(2010225036). |
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| TNF-α regulates netrin-1 expression through enhancing promoter activity |
| BAI Jiu-xu,CAO Ning*,HAN Jing-ming,LI Xu,WANG Dong-hui |
(Department of Blood Purification, General Hospital, PLA Shenyang Military Area Command, Shenyang 110015, Liaoning, China
*Corresponding author.) |
| Abstract: |
| 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. ConclusionTNF-α can regulate netrin-1 expression through modulating netrin-1 promoter activity. |
| Key words: netrin-1 tumor necrosis factor-α transcriptional initiation site |
