低氧对A549细胞糖皮质激素受体表达的影响

Influence of hypoxia on glucocorticoid receptors in A549 cells

目的研究不同低氧时间培养下,人肺腺癌上皮A549细胞糖皮质激素受体α(GRα)和糖皮质激素受体β(GRβ)的表达。方法将A549细胞分成6组,低氧组(3组)和对照组(3组),分别放置于低氧小室(37℃,95%N2,5%CO2)和普通培养箱中(37℃,95%空气,5%CO2)培养。24h、48h、72h后收集细胞,运用倒置显微镜下拍摄照片、RT-PCR和免疫细胞化学方法观察缺氧对A549细胞数目、形态以及细胞内GRα、GRβ mRNA和蛋白表达的影响。结果①低氧组细胞形态发生显著改变,随着低氧时间的延长,细胞生长速度逐渐减慢,细胞肿胀变形逐渐加剧。②RT-PCR的结果显示:24hGRα mRNA表达水平低氧组(0.914±0.079)与对照组(0.883±0.208)比较略有降低,但差异无显著性(P>0.05);48、72hGRα mRNA表达水平低氧组(0.794±0.029、0.828±0.171)与对照组(1.054±0.120、1.187±0.173)比较显著降低,差异有显著性(P<0.05);24、48、72hGRβ mRNA表达水平低氧组(0.439±0.175、0.437±0.117、0.433±0.101)与对照组(0.429±0.140、0.443±0.114、0.553±0.110)比较差异无显著性(P>0.05)。③免疫细胞化学的结果显示:24hGRα蛋白表达水平低氧组与对照组比较无显著改变;48、72hGRα蛋白表达水平低氧组与对照组比较显著降低;24、48、72hGRβ的蛋白表达水平低氧组与对照组比较无显著改变。结论低氧可导致A549细胞GRα的mRNA和蛋白表达水平显著降低;低氧环境可能会影响糖皮质激素作用的发挥。

Objective To study the expression of glucocorticoid receptor α （GRα） and glucocorticoid receptor β （GRβ） under hypoxia conditions in lung cancer A549 cells. Methods A549 cells were divided into six groups, i.e. , three hypoxia groups and three control groups. Both the hypoxia groups and control groups were maintained in hypoxia chamber （37℃ ,95% N2 ,5 % CO2 ） and normoxia incubator（ 37℃ ,95% 空气,5% CO2 ） respectively for 24 h,48 h and 72 h. The mRNA and protein of GRα and GRβ of A549 cells were detected by RT-PCR and immunocytochemistry respectively. Results （ 1 ） There significant morphological alterations were found in A549 cells under hypoxia conditions as compared with the cells grew in the normoxia conditions . （2）The mRNA expression of GRα of 24 h hypoxia groups（0. 914 ±0. 079） was not significantly alterated （P 〉0. 05 ） as compared with that of the control groups（0.883±0. 208）. The mRNA expression of GRα of 48 h and 72 h hypoxia groups （0. 794 ± 0. 029, 0. 828 ± 0. 171 ） were significantly decreased （ P 〈 0. 05 ） as compared with control groups （ 1. 054 ± 0. 120, 1. 187 ± 0. 173）. The mRNA expression of GRβ of 24 h,48 h and 72 h hypoxia groups （0. 439 ±0. 175, 0. 437 ± 0. 117, 0. 433 ± 0. 101 ） were not significantly altered as compared with the control groups （ 0. 429 ± 0. 140, 0. 443 ± 0. 114, 0. 553± 0. 110） （ P 〉 0. 05 ）. （ 3 ） The protein expression of GRα of 24 h group was not significantly altered as compared with the control group. The protein expression of GRα of 48 h and 72 h groups were significantly decreased compared with the control groups. The protein expression of GRβ of 24 h, 48 h and 72 h hypoxia groups were not significantly altered as compared with the control groups. Conclusion The mRNA and protein expression of GRα of A549 cells were decreased under hypoxia condition.