The Transcription Regulation And Molecular Mechanism Of Hsf1 On Tnf-α,hmgb1,il-10 Induced By Burn Serum

Objective:To explore the transcription regulation and molecular mechanism of Heat Shock Transcription Factor 1 (HSF1) on Tumor Necrosis Factor-alpha (TNF-α),High Mobility Group Proteins B1(HMGB1),Interleuldn-10(IL-10) in RAW264.7 induced by burn serum.Methods:1. To construct the eukaryotic expression vector containing mouse Heat Shock Transcription Factor 1 (HSF1) pcDNA3.1-HSF1 after the cDNA of HSF1 was amplified from mouse and the amplified fragment was cloned into pcDNA3.1, the recombinant plasmid was confirmed by restriction enzyme digestion and sequencing comparison.2. The fragment of high mobility group box 1(HMGB1) gene promoter was amplified from mouse genomic DNA by Polymerase Chain Reaction (PCR), the amplified fragment was subsequently cloned into pGL3-basic and constructing the luciferase reporter gene containing mouse HMGB1 wild type promoter, the recombinant plasmid was confirmed by restriction enzyme digestion and sequencing comparison. To construct the luciferase reporter gene containing mouse HMGB1 mutant type promoter after mutating the mouse HMGB1 wild type promoter by overlap extension PCR and the amplified fragment was cloned into pGL3-basic, the recombinant plasmid was confirmed by restriction enzyme digestion and sequencing comparison.3.The mRNA expressions of TNF-α,HMGB1,IL-10 were determined with Sq RT-PCR after pcDNA3.1-HSF1 transfected RAW264.7 induced by burn serum.4. pcDNA3.1-HSFl and the luciferase reporter gene containing mouse TNF-a, HMGB1, IL-10 promoter (wild type+mutant type) and pRL-SV40 transfected RAW264.7 together, then determine and compare the relative luciferase activity of the luciferase reporter gene containing mouse TNF-a, HMGB1, IL-10 wild type promoter and the luciferase reporter gene containing mouse TNF-a,HMGB1, IL-10 mutant type promoter.Results:1. The results of restriction enzyme digestion and sequencing comparison are right, the eukaryotic expression vector containing mouse Heat Shock Transcription Factor 1 (HSF1) pcDNA3.1-HSF1 is constructed successfully.2. The results of restriction enzyme digestion and sequencing comparison are right, the luciferasc reporter gene containing mouse HMGB1 wild type promoter pGL3-HMGB1-Y and the luciferase reporter gene containing mouse HMGB1 mutant type promoter pGL3-HMGB1-T are constructed successfully.3. HSF1 can downregulate the expressions of TNF-α,HMGB1 mRNA and upregulate the expressions of IL-10 mRNA in RAW264.7 induced by bum serum.4. The relative luciferase activity of the luciferase reporter gene containing mouse TNF-α,HMGB 1,IL-10 mutant type promoter is lower significantly than that of the luciferase reporter gene containing mouse TNF-a,HMGB1,IL-10 wild type promoter, P<0.01, there is statistical significance.Conclusions:HSF1 can downregulate the expression of TNF-a,HMGB1 mRNA and upregulate the expression of IL-10 mRNA in RAW264.7 induced by bum serum, which might be affected by the binding of HSF1 to the HSE in the promoter of TNF-a,HMGB 1 and IL-10.