The Research Of AP-2Elements In Regulatory Region Of Hunman Bronchial Epithelial Cells GCLC Gene

BackgroundOxidative stress is one of the many major pathogenesis of acute or chronic lung disease, serious harm to human health. Glutathione (glutathione, GSH) is the body important protective antioxidants. γ-glutamylcysteine synthetase (γ-GCS) is a rate limiting enzyme in GSH synthesis in vivo, consisting of a catalytic subunit (GCLC) and a regulatory subunit (GCLM) components. All GCLC containing γ-GCS substrate binding sites of the catalytic subunit and all, so all of the γ-GCS having catalytic activity. Therefore, GCLC gene expression levels in the body will be affected to a large extent of GSH. Our previous studies analyzed by the transcription factor software analysis found that AP-2transcriptional regulatory elements and NF-κB transcriptional regulatory elements in the human GCLC-790～-766region upstream of the gene. This experiment will be AP-2(-784～-770) binding element, and NF-κB (-790～-785) binding transcriptional regulatory elements for further research to help understand the changes in molecular biology mechanism of GSH.PurposeRole in the analysis of human GCLC-790～-766region upstream of the gene AP-2(-784～-770) components and NF-κB (-790～-785) elements, to explore the possible mechanism of transcriptional regulation. In order to clarify the mechanism of GSH levels in part to provide a theoretical basis for the molecular biological level elucidate the pathogenesis of lung disease.Method1.Design of four kinds of components containing mutant AP-2(-784～-770) gene fragment (-790～-766), and the corresponding synthetic mutant probes. Electrophoretic mobility shift assay (EMSA) experiments to observe whether the mutated probe and nuclear protein capable of specifically binding and detecting a mutated probe super mobility shift assay (Supershift assay) whether the transcription factor AP-2and NF-κB binding to antibody.2.Using overlapping PCR technique AP-2elements (-784～-770) site-directed mutagenesis. And construct a plasmid capable of expressing the mutant luciferase reporter gene.3.The wild-type plasmid, mutant plasmid, respectively in the reference plasmid pRL-TK16HBE cells cotransfected detected after transfection luciferase activity values to determine elements AP-2mutations on human gene transcription activity GCLC.Result1. After testing EMS A and Supershift assay, screening one of the AP-2mutants containing element (-784～-770) gene fragment (-790～-766) and16HBE nuclear protein capable of binding, but not combined the transcription factor AP-2antibody.2. After sequencing, AP-2mutation carriers designed in line with expectations, successfully constructed.3. The mutant was constructed successfully and AP-2in the reference plasmid pRL-TK plasmids were transfected16HBE cells luciferase activity detected value found mutations AP-2(-784～-770) than the wild type plasmid vector fluorescence Luciferase activity was significantly decreased (P<0.01), who described GCLC transcriptional elements AP-2regulatory region (-784～-770) with positive transcriptional regulatory functions.Conclusion1. AP-2transcription factor binding sites, and transcription of the human NF-κB gene transcription start GCLC-790～-766bp upstream of the range.2. AP-2binding element GCLC human gene upstream of the transcription start site (-784～-770) with positive transcriptional regulatory functions.