沉默葡萄糖调节蛋白78基因增强高氧诱导的肺泡上皮细胞凋亡

Effect of glucose regulated protein 78 gene silencing on hyperoxia-induced apoptosis in alveolar epithelial cells

目的探讨小干扰RNA(siRNA)干扰内质网应激相关因子葡萄糖调节蛋白78(GRP78)基因表达对高氧诱导肺泡上皮细胞凋亡的影响及作用机制。方法培养人肺腺癌A549细胞,用构建的siRNA片段通过脂质体LipofectamineTM2000转染A549细胞,将细胞分为高氧空白组、阴性对照(阴性siRNA)组和实验(GRP78-siRNA)组3组,利用950 mL/L O2建立高氧细胞损伤模型。48 h后通过实时定量PCR检测C/EBP同源蛋白(CHOP)mRNA的表达,Western blot技术检测CHOP蛋白表达,流式细胞仪检测细胞凋亡。结果应用siRNA抑制GRP78表达后,GRP78 mRNA和蛋白表达下调、CHOP mRNA和蛋白表达上调、A549细胞凋亡增加。结论下调GRP78基因可增强高氧活化的CHOP凋亡途径,诱导肺泡上皮细胞凋亡。

Objective To investigate the effect and possible mechanism of small interfering RNA（siRNA）-mediated glucose regulated protein 78（ GRP78） gene silencing on hyperoxia-induced apoptosis of alveolar epithelial cells. Methods Human lung adenocarcinoma A549 cells in vitro were transfected with siRNA by LipofectamineTM2000. Cells were divided into untransfected group,scrambled siRNA group and GRP78-siRNA group. A model of hyperoxia-induced cell injury was established by 95% oxygen. After 48 hours,the mRNA and protein levels of GRP78 and C/EBP homologous protein（CHOP）were detected respectively by real-time PCR and Western blotting,and cell apoptosis was measured by flow cytometry. Results By GRP78 siRNA interference,the mRNA and protein levels of GRP78 decreased significantly,while CHOP increased significantly.The apoptosis rate of A549 cells was elevated significantly. Conclusion GRP78 gene silencing may enhance the CHOP apoptotic pathway and promote apoptosis in alveolar epithelial cells induced by hyperoxia,suggesting the potential value of GRP78 as a therapeutic target for the clinical treatment of bronchopulmonary dysplasia.