Investigations of glucocorticoid receptor mutant without ligand binding domain / 医学研究生学报

Objective:To construct expression vector of human glucocorticoid receptor mutant.Methods:cDNA sequence of human glucocorticoid receptor mutant having no LBD was amplificated through RT-nested PCR,PCR product was directionally cloned to pEGFP-N2 plasmid vector,and recombinant plasmids were screened through sequencing.Expressions of recombinant plasmids in human macrophage cell line U937 were observed through fluorescence microscope.Transcriptional activation activity of variant was evaluated through methods of relative activity of luciferase.Results:The 1 601 bp length cDNA was obtained,and result of sequencing confirmed the cloned cDNA sequence is completely coincidence with that of glucocorticoid receptor in Genebank.Expression products of recombinant plasmid mainly located in nucleus.Transcriptional activation activity incresed after recombinant plasmid transfected.Conclusion:Expression vector of glucocorticoid receptor mutant was constructed successfully,which has transcriptional activation activity independent of glucocorticoid.

Regulation of nitric oxide on glucocorticoid receptor function in rat alveolar macrophages treated by LPS in vitro / 第三军医大学学报

Objective To investigate the effects of nitric oxide donor (SNP) on glucocorticoid receptor function in condition of cell inflammation. Methods After fluorescence expression plasmid pGFP-GR transfected rat alveolar macrophges (AMs), nuclear translocation of GFP-GR following to lipopolysaccharide (LPS) and SNP treatment was observed through fluorescence microscope. The transcriptional activation activity of GR was evaluated by the method of relative activity of luciferase. Electrophoretic mobility shift assays (EMSA) were used to measure the activity of NF-?B. Results GFP-GR showed nuclear translocation in 2 h after nitric oxide donor (500 ?mol/L SNP) treatment, and the transcriptional activation activity of GR increased and the activity of NF-?B decreased remarkedly. The phenomena of depressed activity of NF-?B disappeared after the addition of GR special antagonism RU486. Conclusion Nitric oxide donor (500 ?mol/L SNP) exerts anti-inflammatory effect through GR activation in condition of cell inflammation.