Preliminary study on the effect of lipopolysaccharide challenge on the genes expression of inflammatory factors in A549 cells / 中华危重病急救医学

ABSTRACT

Objective To explore the effects of lipopolysaccharide (LPS) on the expression of inflammatory genes in A549 cells line under different concentrations and different action time, this study laid the foundation for further establishment of acute respiratory distress syndrome (ARDS) cell model in the optimal concentration-time way. Methods A549 cells line was incubated routinely in 5%CO2 incubator at 37 ℃ with high glucose DMEM medium which included 10% fetal calf serum. Cells in logarithmic phase was cultured for passage, the cells was count to adjust cell density to (5-7)×105 and tile evenly in six-hole plate. Cells were cultivated for 2 days and once the cells confluence to 50%-60%, serum-free medium DMEM was changed for 12 hours cultivation. 10 mg LPS was added to 10 mL DMEM for oscillated blending to prepare 1 g/L stock solution. 0.5, 1.0 and 2.5 mL LPS stock solution was taken respectively and diluted LPS stock solution for 50 mL constant volume to prepare 0, 10, 20 and 50 mg/L LPS working solution. Then 0, 10, 20 and 50 mg/L LPS solution was added to react for 0, 1, 3 and 5 hours respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to examine mRNA expression of A549 cells line interleukins (IL-6, IL-1β) and tumor necrosis factor-α(TNF-α). LPS action of 10 mg/L for 0 hour was used as the time control group, LPS action of 0 mg/L for 1 hour was used as the concentration control group, and the gene expression was calculated with 2-&Delta;&Delta;Ct method. Results ① As to the time factor, with the same action of LPS concentration, the relative expression levels of inflammatory genes (IL-6, IL-1β and TNF-α) in A549 cells line after being treated with 10 mg/L LPS for 1 hour were significantly higher than those for 0 hour respectively [IL-6 mRNA (2-&Delta;&Delta;Ct) 5.71±0.42 vs. 1.00±0.00, IL-1β mRNA (2-&Delta;&Delta;Ct) 5.63±0.30 vs. 1.00±0.00, TNF-α mRNA (2-&Delta;&Delta;Ct) 5.38±0.61 vs. 1.00±0.00, all P < 0.01], and there were no significant changes in the expression levels of inflammatory genes in A549 cells line of other time groups. ② As to the concentration factor, with the same action time, the relative expression levels of inflammatory genes (IL-6, IL-1βand TNF-α) in A549 cells line after being treated with 10 mg/L LPS for 1 hour were significantly higher than with 0 mg/L for 1 hour respectively [IL-6 mRNA (2-&Delta;&Delta;Ct) 5.70±0.64 vs. 1.00±0.00, IL-1β mRNA (2-&Delta;&Delta;Ct) 6.25±0.25 vs. 1.00±0.00, TNF-α mRNA (2-&Delta;&Delta;Ct) 5.57±0.25 vs. 1.00±0.00, all P < 0.01], there were no significant changes in the expression levels of inflammatory genes in A549 cells line of other concentration groups. Conclusion The LPS concentration of 10 mg/L and the action time of 1 hour are the most suitable concentration-time conditions for establishing ARDS cell models of A549 cells line.