双荧光素酶报告质粒检测小鼠巨噬细胞热休克转录因子1调控核转录因子kappa B活性的研究

A Study of the Effects of HSF1 on NF-κB Activity in RAW264.7 Macrophages by Dual-luciferase Assay

目的:观察烧伤血清刺激后小鼠巨噬细胞NF-κB活性的变化,以及HSF1对NF-κB可能的凋控作用。方法:制作15%TBSAⅢ°烧伤小鼠模型,提取烧伤血清通过表达质粒与报告质粒共转染,检测烧伤血清诱导下NF-κB活性的变化以及过表达HSF1后NF-κB活性的变化规律结果:对比正常血清,烧伤血清刺激后相时荧光素酶活性早期即明显增加(p<0.05),这种变化在诱导后2 h即达到高峰,12 h后逐渐下降;过表达HSF1可以显著抑制烧伤血清引起这种活性变化(P<0.05)。结论:烧伤后NF-κB早期即活化,热休克反应可能通过HSF1途径抑制NF-κB的活性。

Objective：To study the effects of heat shock factor 1 （HSF1） on nuclear factor kappa B （NF-κB） activity induced by burns serum. Methods：HSFl expression plasmid was first constructed and identified. Burns serum was extracted from burned animal models. Then NF-κB reporter plasmid was transfected into mouse RAW264.7 macrophages, stimulated by nomal or burns serum at different time, and the luciferase activity was detected by dual-luciferase assay. Subsequently, the reporter plasmid and HSF1 gene was cotransfeeted into maerophages, cultivated with burns serum for 2 hours, then the cells were schizolysised and detected by dual-lueiferase assay. Results：The luciferase activity increased remarkably after burns serum stimulation at early time, and reached peak 2 hours ofter the burn, but this change was inhibited after HSF1 overexpression. Conclusion：After bums serum stimulation NF-κB was significantly activated. HSF1 gene may down-regulates the NF-κB transcription activity afterbumed.