糖皮质激素诱导人气道上皮细胞9HTE_0凋亡的研究

Glucocorticoid-induced apoptosis of human airway epithelial 9HTE_0 cells

目的探讨不同浓度及不同时间的糖皮质激素诱导人气道上皮细胞9HTE0凋亡的机制。方法采用Annexin V/PI双染法检测低浓度组(0.3μmol/L)、中浓度组(3μmol/L)、高浓度组(10μmol/L)地塞米松(dexamethasone,Dex)作用6 h、12 h、24 h对人气道上皮细胞9HTE0早期凋亡的比例变化;RT-PCR法检测凋亡相关基因Bcl-2、Bax mRNA的表达情况;细胞免疫荧光法检测Bcl-2、Bax蛋白的定位及表达情况。结果 Annexin V/PI双染法检测中、高浓度组Dex在作用12 h、24 h后,早期凋亡率较正常组有显著性差异(P<0.05);Bcl-2 mRNA于高浓度组Dex作用6 h后即出现变化,而在中、高浓度组Dex作用12 h及24 h后,Bcl-2 mRNA均显著下降(P<0.05),Bax mRNA均显著增高(P<0.05);Bcl-2、Bax蛋白定位于细胞核和细胞浆中,中、高浓度组Dex作用24 h后荧光有明显变化。结论糖皮质激素作为哮喘一线用药,诱导了人气道上皮细胞9HTE0凋亡同时使其具有浓度和时间依赖性,从而为气道上皮的损伤提供了理论依据。

To explore the mechanisms human airway epithelial 9HTE0 cell apoptosis induced by glucocorticoid at different concentrations and times.Annexin V/PI double staining was used to detect early apoptosis proportion of 9HTE0 cell treated with 0.3 μmol/L（low concentration group）,3 μmol/L（middle concentration group）,and 10 μmol/L（high concentration group） dexamethasone for 6 h,12 h,and 24 h,respectively.We found that the early apoptosis proportion of middle and high concentration groups were significantly different from that of control group after 12 h and 24 h treatment（P 0.05）.RT-PCR showed that Bcl-2 mRNA of high,middle and high concentration groups decreased significantly at 6 h at 12 h and 24 h respectively as compared with control group（P 0.05）,while Bax mRNA of middle and high concentration groups increased significantly at 12 h and 24 h respectively as compared with control group（P0.05）.Immunofluorescence demonstrated that Bcl-2 and Bax protein were expressed in cell nucleus and cytoplasm,and the fluorescence of middle and high concentration groups had remarkable changes at 24 h.the result indicate that glucocorticoid as the fist line treatment could induce apoptosis of human airway epithelial 9HTE0 cell in time and concentration dependent manner,while provide a theoretical basis for damage of airway epithelium.