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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 of small interfering RNA (siRNA) on the expression of ER stress-related factor glucose regulated protein 78 (GRP78) on the apoptosis of alveolar epithelial cells induced by hyperoxia and its mechanism. Methods Human lung adenocarcinoma A549 cells were cultured and transfected into A549 cells with LipofectamineTM 2000 by using the constructed siRNA fragment. The cells were divided into three groups: high oxygen blank group, negative control group (GRP78-siRNA) and negative control group , A hyperoxic cell injury model was established using 950 mL / L O2. After 48 h, the expression of C / EBP homologous protein (CHOP) mRNA was detected by real-time quantitative PCR, the expression of CHOP protein was detected by Western blot, and the apoptosis was detected by flow cytometry. Results After siRNA inhibited the expression of GRP78, the expression of GRP78 mRNA and protein were down-regulated, the expression of CHOP mRNA and protein was up-regulated, and the apoptosis of A549 cells was increased. Conclusion Down-regulation of GRP78 can enhance the pathway of CHOP apoptosis induced by hyperoxia and induce the apoptosis of alveolar epithelial cells.