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为探讨热休克因子1(heatshockfactor 1,HSF1)活化和过表达对内毒素(endotoxin ,ET)所致粒细胞集落刺激因子(granulocyte colonystimulatingfactor,G CSF)基因表达的影响,采用大肠杆菌内毒素即脂多糖(lipopolysaccharide ,LPS)处理RAW2 6 4 7巨噬细胞,并通过热休克预处理诱导HSF1活化,采用Western印迹检测HSP70的表达观察HSF1的活化情况,RT PCR检测热休克反应(heatshockresponse ,HSR)对G CSFmRNA表达的影响;构建HSF1的pcDNA3 1真核表达质粒,采用脂质体转染法建立HSF1过表达RAW 2 6 4 7巨噬细胞株,用免疫细胞化学和Western印迹观察HSF1的表达,RT- PCR及Northern印迹进一步研究HSF1对G CSF基因表达的可能影响.发现LPS诱导巨噬细胞中G- CSFmRNA表达增多,并随时间的延长,表达量逐渐增加;与单纯内毒素处理组相比,热休克预处理后,LPS诱导的巨噬细胞G- CSFmRNA的表达明显被抑制;建立的稳定表达HSF1的RAW 2 6 4 7细胞株中有HSF1蛋白的核移位;HSF1过表达可明显抑制LPS诱导的RAW2 6 4 7巨噬细胞G -CSFmRNA的表达.上述结果表明热休克预处理能抑制LPS诱导的巨噬细胞G- CSFmRNA的表达;HSF1过表达可抑制内毒素诱导的巨噬细胞G CSFmRNA的表达.
To investigate the effect of heat shock factor 1 (HSF1) activation and overexpression on the gene expression of granulocyte colony stimulating factor (granulocyte colony stimulating factor, G CSF), endotoxin of E.coli RAW2647 macrophages were treated with lipopolysaccharide (LPS) and activated by heat shock pretreatment. The expression of HSP70 was detected by Western blotting to observe the activation of HSF1. RT-PCR was used to detect the heat shock response (HSR) G CSFmRNA expression. HSF1 pcDNA3 1 eukaryotic expression plasmid was constructed. HSF1 overexpression RAW2647 macrophage cell line was established by lipofection. The expression of HSF1 was observed by immunocytochemistry and Western blot. RT - PCR and Northern blotting to further study the possible effect of HSF1 on the expression of G CSF gene.It was found that the expression of G-CSF mRNA in LPS-induced macrophages increased and over time, the expression of G-CSF mRNA increased gradually.Compared with the endotoxin-treated group, After pretreatment with heat shock, the expression of G-CSF mRNA in LPS-induced macrophages was significantly inhibited. The HSF1-stably transfected RAW2647 cells with HSF1 protein HSF1 overexpression could significantly inhibit G-CSF mRNA expression induced by LPS in RAW2647 macrophages.The above results show that heat shock preconditioning can inhibit the LPS-induced G-CSF mRNA expression in macrophages and over-expression of HSF1 Inhibit endotoxin-induced macrophage G CSF mRNA expression.