Study On The Protective Role Of HSF1 In Inflammatory Response After Burned In Mouse Macrophage Cells

CHAPTER 1Construction and Identification of Mouse IL-10 and TNF-αpromotor luciferase report gene vectorObjective:To construct the mouse IL-10 and TNF-αpromotor luciferase report gene vector.Methods:The mouse IL-10 and TNF-αpromoter from RAW264.7 macrophage cells DNA was amplified by PCR, and was inserted into the luciferase report gene pGL3-basic vector to form plasmid(wild type). Mutated mouse IL-10 and TNF-αpromoter was prepared by PCR amplified of wild type plasmid DNA with site-directed mutated primers. Then mutated DNA fragments were also ligated into pGL3-basic vector to form plasmid(mutated type). The amplified DNA sequence were both confirmed by restriction endonuclease digestion and sequencing.Results:Restriction endonuclease digestion showed 680bp,240bp DNA fragments of the IL-10 and TNF-αpromoter. The sequencing results indicated that the amplified sequence was the same as Gene Bank shows, the core sites of IL-10 and TNF-αpromoter("ttctggaa","GAA") were replaced by "tactgccc","GCC".Conclusion:The mouse IL-10 and TNF-αpromoter luciferase reporter gene vector has been constructed successfully. CHAPTER 2Construction of Expression System of NF-κB p65 subunit of MouseObjective:To construct the mouse NF-κB p65 subunit expression plasmids.Methods:The cDNA encoding NF-κB p65 was isolated by using RT-PCR method from the mouse RAW264.7 macrophage cells. P65 subunit was then cloned into vector pcDNA3.1(-), and identified by restriction enzyme analysis and sequencing.Results:A 1664bp DNA fragment was obtained by RT-PCR, then was cloned into pcDNA3.1(-). The sequencing showed two samesense mutations compaired with GeneBank, but there is no difference in translation.Conclusion:The construction of an expression system of NF-κB p65 subunit was constructed, which established the foundation for the research on the role of NF-κB post-burned. CHAPTER 3Study of Effects of HSF1 on NF-κB Activity in RAW264.7 Macrophages Induced by Burns SerumObjective:To study the effects of heat shock factorl (HSF1) on nuclear factor kappa B (NF-κB) activity induced by burns serum.Methods:HSF1 expression plasmid was first constructed and identificated. 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 with different time, and the luciferase activity was detected by dual-luciferase assay. Subsequently, the reporter plasmid and HSF1 gene was cotransfected into macrophages, cultivated with burns serum for 2 hours, then the cells were schizolysised and detected by dual-luciferase assay.Results:The luciferase activity increased remarkably after burns serum stimulation at early time, and reached peak after 2 hours of effection, but this change was inhibited after HSF1 overexpression.Conclusion:After burns serum stimulation NF-κB was significantly activated. HSF1 gene may down-regulates the NF-κB transcription activity after burned. CHAPTER 4Effects of Interaction of HSF1 and NF-κB on Activity of TNF-α,IL-10 and HMGB1 Promoter Induced by Burns Serum in RAW264.7 MacrophagesObjective:To study the effects of interaction of HSF1 and NF-κB on activity of inflammation factors promoter induced by burns serum.Methods:Expression plasmid pcDNA3.1(-)/HSF1 and pcDNA3.1(-)/p65 were cotransfected into mouse RAW264.7 macrophages with inflammation factors promoter luciferase report gene vectors. Then stimulated by burns serum for 2 hours, and the luciferase activity was detected by dual-luciferase assay.Results:The luciferase activity of TNF-a,HMGB1, IL-10 promoter increased remarkably when HSF1 was overexpression, and respectively raised 1.92-fold,2.62-fold and 1.5-fold. But after HSF1 and p65 both overexpression their activity respectively raised 1.29-fold,2.74-fold and 1.13-fold, and the activity of TNF-a and IL-10 promoter showed difference when compaired with only HSF1 overexpression. Activity of inflammation factors promoter increased when macrophages stimulated with higher concentration of burn serum.Conclusion:HSF1 and NF-κB may interact on activity of TNF-a and IL-10 promoter after burned. Activity of TNF-a,HMGB1 IL-10 promoter increased in more serious burns.