| BackgroundHypoxia is a common pathophysiologie processes concomitant with respirtory disease, for example,asthma and chronic obstructure pulmonary disease(COPD).hypoxia is a potent stimulus for iflammation and remodline of airway.Glucocortieoid are the most common and effective drugs for respirtory diseases,glucocorticoids bind to glucocorticoid receptors in the cytoplasm which then dimerize and translocate to the nucleus,where they bind to glucocorticoid response elements(GRE) on glucocorticoid-responsive genes,resulting in increased transcription.Glucocorticoids may inerese anti-inflammatory proteins,including lipoeortin-1,interleukin-10, interleukin-1 receptor antagonist and neutral endopeptidase.The most striking effect of glucocorticoids is to inhibit the expression of multiple inflammatory genes(eytokines, enzymes,receptors and adhesion molecules).It is more likely to be due to a direct inhibitory interaction between activated glucocorticoid receptors and activated transcription factors,such as nuclear factor-kappa B and activator protein-1,which regulate the inflammatory gene expression.Glucocorticoid receptor(GR) gen can be mainly transeripted to two isoforms,GRaand GRfβ,GRa was recognized as the "classic GR" which mediated glucoeorticoid action.GRβhas been found to act as a dominant negative inhibitor of GRa-mediated transactivation in vitro.A small proportion of patients with asthma or COPD fail to respond sensitively even resist to high doses of glucocorticoids,However,the debilitating side effects of high-dose glucocorticoid therapy militate against its use in ever-increasing doses.the underlying molecular mechanisms for the glucocorticoid resistant are not fully understood.but there is not intemal study show if hypoxia is one of the mechenisms of glucocordticoid resistance.this study is to show this by study the expression of GRaand GRβunder hypoxia conditions in lung cancer A549 cells.ObjectiveThis study was designed to study the expression of GRa and GRβunder hypoxia conditions in lung cancer A549 cells and analyse the relationship of hypoxia and glucocorticoid resistance.Methods1.We cultured A549 cells for 24h,48h and 72h at hypoxia chamber and normoxia conditions respectively.2.We invsetigate the alterations of morphological and the number of cells cultured at hypoxia chamber compared with the cells cultured at normoxia conditions for 24h,48h and 72h by inverted microscope.3.We study the expression of GRaand GRBmRNA under hypoxia conditions in lung cancer A549 cells compared with the cells cultured at normoxia conditions for 24h,48h and 72h by RT-PCR.4.We study the expression of GRaand GRβprotein under hypoxia conditions in lung cancer A549 compared with the cells cultured at normoxia conditions for 24h,48h and 72h by immunocytochemistry.Results1.The number and morphological alterations of cells cultured at hypoxia chamber compared with the cells cultured at normoxia conditions for 24h,48h and 72h.There were significant morphological alterations in A549 ceils under hypoxia conditions as compared with the cells grew in the normoxia conditions for 48h and 72h. The 24h hypoxia groups didn't change obviously compared with normoxia groups. The number of cells cultured at hypoxia chamber compared with the cells cultured at normoxia conditions for 24h didn't change obviously.But the number of cells cultured at hypoxia chamber for 48h reduced at 50%of that cultured at normoxia conditions.The number of cells cultured at hypoxia chamber for 72h reduced at 25%of that cultured at normoxia conditions.2.The mRNA expression of GRaand GRβof 24h,48h and 72h hypoxia groups compared with that of the control groups.The mRNA expression of GRaof 24h hypoxia groups(0.914±0.079) was not significantly alterated(P＞0.05)as compared with that of the control group (0.883±0.208).The mRNA expression of GRaof 48h and 72h hypoxia groups (0.794±0.029,0.828±0.171)were significantly decreased(P＜0.05) as compared with that of control groups(1.054±0.120,1.187±0.173).The mRNA expression of GRβof 24h,48h and 72h hypoxia groups(0.439±0.175,0.437±0.117,0.433±0.101)were not significantly alterated as compared with that of the control groups(0.429±0.140,0.443±0.114,0.553±0.110)(P＞0.05).3.The protein expression of GRaand GRβof 24h,48h and 72h hypoxia Groups compared with that of the control groups.The protein expression of GRaof 24h hypoxia group was not significantly alterated as compared with the control group.The protein expression of GRaof 48h and 72h groups were significantly decreased as compared with the control groups.The protein expression of GRβof 24h,48h and72h hypoxia groups were not significantly alterated as compared with the control groups.ConclusionsThe expression of the mRNA and the protein in A549 cells at hypoxia chamber for 48h and 72h are decreased obviously as compared with the control groups.Hypoxia is maybe one of the mechenisms of glucocorticoid resisitant.
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