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目的探讨缺氧时乳腺癌细胞(MDA-MB-435细胞)肌动蛋白细胞骨架和细胞黏附能力改变与RhoA-ROCK信号通路的关系。方法对体外培养的MDA-MB-435细胞进行缺氧16h处理,或在缺氧前用ROCK的特异性抑制剂Y-27632(10μm)预处理1h,再进行缺氧处理。用光学显微镜观察MDA-MB-435细胞形态的改变;用FITC标记的鬼笔环肽标记F-肌动蛋白,用流式细胞仪技术分析肌动蛋白细胞骨架的重组情况;噻唑蓝比色法(MTT)检测细胞对人工重组基底膜(Matrigel)的黏附能力。结果乳腺癌细胞经16h缺氧处理,细胞内F-肌动蛋白含量和细胞形态发生明显改变;MDA-MB-435细胞对人工重组基底膜的黏附能力明显增强;预先使用Y-27632再进行缺氧处理,可防止缺氧所致乳腺癌细胞内肌动蛋白细胞骨架以及细胞形态的改变,同时其对人工重组基底膜的黏附能力亦得以恢复。结论RhoA-ROCK信号通路可能参与缺氧所致乳腺癌细胞肌动蛋白细胞骨架的重组和黏附能力的改变。
Objective To investigate the relationship between the changes of actin cytoskeleton and cell adhesion and the RhoA-ROCK signaling pathway in hypoxia-induced breast cancer cells (MDA-MB-435 cells). Methods MDA-MB-435 cells cultured in vitro were treated with hypoxia for 16 hours or pre-hypoxia with ROCK-specific inhibitor Y-27632 (10μm) for 1 hour before hypoxia. The morphological changes of MDA-MB-435 cells were observed with light microscopy. F-actin was labeled with FITC-labeled phalloidin and the actin cytoskeleton was analyzed by flow cytometry. Thiazolyl blue colorimetry (MTT) was used to detect the adhesion of cells to Matrigel. Results After 16h hypoxia treatment, the content of F-actin and cell morphology in breast cancer cells were significantly changed. The adhesion capacity of MDA-MB-435 cells to artificial recombinant basement membrane was significantly enhanced. Pre-treatment with Y-27632 Oxygen treatment can prevent hypoxia-induced changes in actin cytoskeleton and cell morphology in breast cancer cells, and its adhesion to artificial recombinant basement membrane is also restored. Conclusion The RhoA-ROCK signaling pathway may be involved in the reorganization and adhesion of actin cytoskeleton in hypoxia-induced breast cancer cells.