甲状腺素对人气道上皮细胞黏蛋白表达的调控

Thyroid hormone regulates mucin gene expression and its mechanism in human airway epithelial cells

目的研究甲状腺素对气道黏液分泌的影响及其调控机制。方法体外培养肺腺癌细胞株A549,给予甲状腺素刺激,用逆转录-多聚酶联反应法(RT-PCR)检测干预前后细胞中黏蛋白(MUC)5AC mRNA含量,酶联免疫吸附测定法(ELISA)检测培养上清中MUC5AC蛋白含量;用DNA重组技术,构建含荧光素酶报告基因和MUC5AC启动子不同长度片段的嵌合质粒,转染A549细胞,检测其荧光素酶的比活性。结果甲状腺素能够显著抑制MUC5AC基因转录和蛋白表达(t=5.35,P<0.05),并能使含有启动子序列-1330/+48bp、-1250/+48bp的嵌合质粒表达荧光素酶减少,与对照组相比差异有显著性(分别为t=3.08,P<0.05和t=5.00,P<0.05),但不能减少含有-1200/+48bp和-1020/+48bp的嵌合质粒荧光素酶表达(分别为t=0.82,P>0.05和t=0.40,P>0.05)。结论甲状腺素能够抑制MUC5AC的基因转录和蛋白表达,MUC5AC基因5'上游-1.25/-1.20kb区域存在参与甲状腺素调控该基因表达的调控元件。

[Objective] To investigate the effect of thyroid hormone on airway mucous secretion and its mechanism. [Methods] The lung adenoearcinoma cells of the line A549 were cultured and divided into 2 groups： control group and T3 group. Cells were cultured in serum-free medium containing all-trans-RA either with or without thyroid hormone. The muein （MUC） 5AC mRNA level was analyzed by RT-PCR, MUC5AC protein was detected by enzyme-linked immunosorbent assay, Fragments ranging in size from 1.33 to 1.02 kb adjacent to the 5＇transcriptional start site of MUC5AC were cloned into a lueiferase reporter vector-pGL-3 enhancer vector, constructs were transfeeted into A549 cells, MUC5AC promoter activity was determined by measuring luciferase activity. [Results] The MUC5AC mRNA expression and protein expression of T3 group were （0.19±0.02） and （0.14±0.04） respectively, both significantly lower than those of the control group [（0.40±0.04） and （0.24±0.02）, both P 〈0.05]. The luciferase activity in A549 ceils transfeeted with the 1.33 and 1.25 kb constructs were （6.5±1.6） and （6.2±0.7） respectively, both significantly lower than those of the control groups [（12.5±4.2） and （11.5±2.4）, both P 〈0.05], but the lueiferase activity in A549 cells transfected with the 1.2 and 1.02 kb constructs were not suppressed significantly. [Conclusion] T3 suppresses MUC5AC expression and its fragment of 1.20 to 1.25 kb adjacent to the 5＇ transcriptional start site is important to the regulation by thyroid hormone.