氧化应激诱导肺泡Ⅱ型上皮细胞凋亡时NF-κB和Bak的表达变化

Expression of NF-κB and Bak in oxidative stress-induced apoptosis of alveolar type Ⅱ epithelial cells

目的:探讨氧化应激状态下肺泡Ⅱ型(alveolar typeⅡ,ATⅡ)上皮细胞的凋亡情况及核因子-κB(nuclear factor-κB,NF-κB)p65和Bak表达变化。方法:原代培养大鼠ATⅡ细胞,用过氧化氢(hydrogen peroxide,H2O2)500μmol/L处理不同时间,建立氧化损伤性细胞模型。相差显微镜下观察细胞形态变化。流式细胞仪检测H2O2刺激后细胞凋亡率的变化。蛋白质免疫印迹法(Western blot)检测ATⅡ细胞受H2O2刺激后NF-κB p65和Bak的表达变化。免疫荧光染色后,激光共聚焦显微镜下观察NF-κB p65的变化和细胞内分布情况。结果:受H2O2刺激3 h后,ATⅡ细胞体积减小,胞内颗粒数目减少,细胞核固缩。与正常对照组比较,受H2O2刺激1 h和3 h后,ATⅡ细胞的凋亡率明显上升(P=0.000,P=0.000)。Western blot检测发现H2O2刺激3 h后,细胞核内NF-κB p65的表达明显增加(P=0.000),ATⅡ细胞Bak蛋白的表达增加(P=0.000)。激光共聚焦显微镜观察发现正常细胞NF-κB p65表达微弱,H2O2刺激3 h后,NF-κB p65表达明显增强,主要集中分布于细胞核。结论:氧化应激能诱导ATⅡ细胞凋亡,在此过程中NF-κB p65在细胞核内的表达增加,Bak的表达也增加,两者参与了ATⅡ细胞的凋亡。

Objective：To investigate the changes of nuclear factor-κB（NF-κB）p65 and Bak expression during the apoptosis of typeⅡ（ATⅡ）epithelial cells induced by oxidative stress,and the apoptotic outcomes of ATⅡ cells as well. Methods：Primary ATⅡ cells were separated,purified and cultured from adult Sprague-Dawley rats treated with 500 μmol/L hydrogen peroxide（H2O2）for different periods of time,with the purpose of establishing an apoptotic cell model by oxidative injuries. The change of cell morphology was explored by phase contrast microscope. Apoptotic ratio was measured by flow cytometry. The changes in expression of NF-κB p65 and Bak were investigated by Western blot. The location of NF-κB p65 was detected by immunofluorescence and observed under laser confocal microscopy. Results：After exposure to H2O2 for 3 h,ATⅡ cells turned smaller,with karyopycnosis and reduction of cytoplasmic granules. The cell apoptotic ratios after exposure to H2O2 for 1 h and 3 h were both increased obviously（P=0.000,P=0.000）,compared with those of normal control group. Western blot detected that the nucleic expression of NF-κB p65 and the expression of Bak after exposure to H2O2 for 3 h were increased remarkably compared with those of normal control group（P=0.000,P=0.000）. The nucleic translocation of NF-κB p65 after H2O2 treatment was also increased in the merged images under laser confocal microscopy. Conclusion：High level of oxidative stress induces ATⅡ cells apoptosis and up-regulates the nuclei expression of NF-κB p65 and the expression of Bak.