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.
Abstract
To explore the effect of heat shock factor 1(HSF1) on expression alteration of granulocyte-colony stimulating factor(G-CSF) in RAW264.7-macrophage,RAW264.7-macrophge were pretreated with heat shock(42℃,1 hour) to activate production of HSF1 and followed by treatment with lipopolysaccharide(LPS) at dose of 800 mg/ml.Western blot analysis revealed that heat treatment of the cells led to production of HSF1 from macrophage,being started at 4 hours reached a peak at 12 hour and lasted for 36 hour.LPS alone induced increase of G-CSF mRNA level and this enhancement was declined after heat shock pretreatment as demonstrated by RT-PCR assay.To further confirm the suppressive effect of HSF1 on G-CSF1 expression,an eukaryonic expression vector pcDNA3.1/myc-His(-)B/HSF1 was constructed and stable expression colony also selected following transecting into RAW 264.7-macrophage;Western blot analysis showed over expression of HSF1 protein of 72 kD in transfection cells which was proved by immunostaining.Furthermore,high concentration of HSF1 in turn repressed the LPS-induced up-regulation of G-CSF transcript in RAW264.7-macrophage.These results provide further evidences of the repressive role of HSF1 in LPS-induced up-regulation of G-CSF in macrophage.
关键词
热休克因子1 /
RAW264.7巨噬细胞 /
内毒素 /
粒细胞集落刺激因子 /
热休克反应
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Key words
HSF1 /
RAW264.7 macrophages /
lipopolysaccharide /
endotoxin /
G-CSF /
heat shock response
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