Effects Of Gliotoxin In The Vitro Model Of Human Bronchial Epithelial Cell Infected By Aspergillus Fumigatus

Objective According to establishing an in vitro model of human bronchial epithelialcell（HBE） infected by Af, we sought to determine effects of its potential mechanismsof GT that are involved in human bronchial epithelial cell infected by Af.Methods1. To observe the role of of GT on the expression of human bronchialepithelial cell apoptosis and Intercellular adhesion molecule-1（ICAM-1）.1) toxinpreparation. GT was dissolved in a95%ethanol solution and then, this solution wasdeveloped to permit sufficient solubility to reach concentration around the TC50valueusing DMEM;2) HBE preparation. HBE were seeded in9cm dishes, covered with10ml of medium per dish and allowed to grow to confluence for48h at37℃and5%CO₂;3) adding toxin. GT was prepared for3concentrations using DMEM. Whenthe confluence over each well in the plate was approximately90%, the mediumincluding GT were added to each well and then allowed to grow for24h at37℃and5%CO₂。3. Results quantification. After HBE were exposed to GT for24h, Cells weretrypsinized centrifuged for5min ar1000rpm, and the pellet was washed with PBS.Assessment of apoptosis and ICAM-1of HBE were confirmed by real-time PCR andannexin V staining.2. an in vitro model of human bronchial epithelial cell（HBE）infected by Af was established.1) Af strains and growth media. Af293that were usedfor the corresponding genome sequencing projects of each fungal species, is preparedon Potato dextrose agar plate.2).HBE preparation. HBE were seeded in9cm dishes,covered with10ml of medium per dish and allowed to grow to confluence for48h at37℃and5%CO₂.3)The vitro model. Af conidia were harvested from3days oldcultures on Potato dextrose agar plates by flooding the surface of the plates with5mlof PBS containing0.025%（v/v） Tween-20and rocking gently. They were washedtwice in phosphate-buffered saline（PBS） and resuspended in the DMEM medium witha final concentration of105conidia/ml. The conidia were inoculated in the dishes including HBE, which were incubated at37℃and5%CO₂for2h to let the fungusadhere to the cell surface. To remove nonattached conidia, the culture was washed,new DMEM medium was added and the culture was incubated at37℃and5%CO₂for12h,24h and36h.4). Results quantification. Assessment of apoptosis by flowcytometric analysis. The expressions of GT secreted by Af and apoptosis and ICAM-1of HBE were confirmed by real-time PCR.Results1. Realtime-PCR analysis revealed that the expressions of the apoptosis genes Bakand Bax both were up-regulation after HBE were exposed to GT（p<0.005å'Œp<0.05）,especially the gene Bak was obvious.2. Realtime-PCR analysis revealed that expression of secondary metabolite GT at12hand24h time point have no statistical significance compared with control（p>0.05）.However, the expression of GT at36h time point increase obviously（p<0.05）.3. Realtime-PCR analysis revealed that the expressions of the apoptosis genes werebeginning to increase from24h time point, to reach a peak at36time point（p<0.05）,especially the gene Bak was obviously up-regulation（p<0.005）. Meanwhile, Bcl-2expressed down-regulation at36time point（p<0.05）.Conclusion1. We demonstrate that GT may be an important role in the model of HBE infected byAf.2. We demonstrate that GT may kill HBE by inducing cell apoptosis in the model.3. We demonstrate that GT may affect the immune response of HBE at someconcentration.