脂多糖处理对A549细胞炎性因子基因表达的影响

Preliminary study on the effect of lipopolysaccharide challenge on the genes expression of inflammatory factors in A549 cells

目的探讨不同浓度不同作用时间下脂多糖(LPS)对A549细胞炎性因子基因表达的影响,为进一步确立急性呼吸窘迫综合征(ARDS)细胞模型的最适浓度-时间条件奠定基础。方法取A549细胞,用含10%胎牛血清的高糖DMEM培养基于37?℃、5%CO2培养箱常规培养,取对数生长期的A549细胞进行传代,计数细胞并调整细胞密度为(5~7)×105个,于6孔板中培养2d,当细胞融合至50%~60%时换无血清培养基DMEM培养12h。取10mgLPS加入10mLDMEM培养基振荡混匀,配制1g/L储存液;取0.5、1.0、2.5mL的LPS储存液用DMEM溶液稀释定容至50mL,配制成0、10、20、50mg/L的LPS工作液。然后以0、10、20、50mg/L的LPS分别作用0、1、3、5h,采用反转录-聚合酶链反应(RT-PCR)检测细胞白细胞介素(IL-6、IL-1β)、肿瘤坏死因子-α(TNF-α)的mRNA表达,以10mg/L的LPS作用0h为时间对照组,以0mg/L的LPS作用1h为浓度对照组,采用2^-ΔΔCt法计算基因表达量。结果①时间因素方面,在相同LPS浓度作用下,A549细胞经10mg/L的LPS作用1h时IL-6、IL-1β、TNF-α的mRNA表达水平均明显高于0h时〔IL-6mRNA(2^-ΔΔCt):5.71±0.42比1.00±0.00,IL-1βmRNA(2^-ΔΔCt):5.63±0.30比1.00±0.00,TNF-αmRNA(2^-ΔΔCt):5.38±0.61比1.00±0.00,均P<0.01〕,其他时间点间细胞炎性因子基因表达水平均无明显改变。②浓度因素方面,在相同作用时间下,A549细胞经10mg/L的LPS作用1h时IL-6、IL-1β、TNF-α的mRNA表达水平均明显高于0mg/L的LPS作用1h时〔IL-6mRNA(2^-ΔΔCt):5.70±0.64比1.00±0.00,IL-1βmRNA(2^-ΔΔCt):6.25±0.25比1.00±0.00,TNF-αmRNA(2^-ΔΔCt):5.57±0.25比1.00±0.00,均P<0.01〕,其他浓度LPS组间炎性因子基因表达水平均无明显改变。结论10mg/L的LPS作用A549细胞1h为建立ARDS细胞模型的最适作用浓度-时间条件。

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^-ΔΔCt 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^-ΔΔCt): 5.71±0.42 vs. 1.00±0.00, IL-1β mRNA (2^-ΔΔCt): 5.63±0.30 vs. 1.00±0.00, TNF-α?mRNA (2^-ΔΔCt): 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^-ΔΔCt): 5.70±0.64 vs. 1.00±0.00, IL-1β mRNA (2^-ΔΔCt): 6.25±0.25 vs. 1.00±0.00, TNF-α mRNA (2^-ΔΔCt): 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.