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目的研究氧化应激条件下中间纤维蛋白细胞角蛋白(cytokeratin)的抗凋亡作用。方法利用RNA干扰(RNAi)技术敲低细胞内细胞角蛋白18(CK18)的表达,免疫印迹法检测细胞角蛋白的表达水平,并通过A549细胞建立细胞角蛋白敲低稳定细胞系。利用免疫荧光染色技术观察细胞角蛋白对细胞骨架的影响。利用流式细胞技术检测细胞角蛋白对细胞内ROS(reactive oxygen species)水平及氧化应激诱导细胞凋亡的影响。结果筛选获得3个干扰效果较好的RNAi序列,成功利用A549细胞建立细胞角蛋白敲低的稳定细胞系(A549-KD)。CK18表达敲低导致细胞内多种细胞角蛋白表达广泛下调,并破坏细胞微丝及微管骨架结构。氧化应激条件下A549-KD细胞抗凋亡活性显著降低。结论细胞角蛋白参与维持细胞骨架结构,对抗ROS诱导的细胞凋亡,提示其在细胞的氧化应激过程中发挥保护作用。
Objective To investigate the anti-apoptotic effect of cytokeratin on oxidative stress. Methods The expression of cytokeratin 18 (CK18) was knocked down by RNA interference (RNAi), the cytokeratin expression was detected by Western blotting, and the cytokeratin knockdown stable cell line was established by A549 cells. The effect of cytokeratin on cytoskeleton was observed by immunofluorescence staining. The effects of cytokeratin on reactive oxygen species (ROS) level and apoptosis induced by oxidative stress were detected by flow cytometry. Results Three RNAi sequences with high interference were screened and A549 cells were successfully used to establish a stable cell line (A549-KD) that knocked down cytokeratin. Knockdown of CK18 results in the widespread down-regulation of multiple cytokeratin expression in cells and disrupts the microfilament and microtubule cytoskeleton of cells. The anti-apoptotic activity of A549-KD cells was significantly decreased under oxidative stress. Conclusion Cytokeratin is involved in maintaining the cytoskeleton structure and antagonizing ROS-induced apoptosis, suggesting that cytokeratin plays a protective role in cellular oxidative stress.