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目的在缺氧微环境下观察微管相关蛋白4(microtubule-associated protein 4,MAP4)在人永生化角质形成细胞株(HaCaT细胞株)中的表达变化及对细胞迁移能力的影响。方法利用Western blot检测早期缺氧后(1%O2,缺氧时间分别为0.5、1、3 h)MAP4在HaCaT细胞中的表达变化;设立正常细胞组、干扰MAP4组及阴性对照组(空病毒转染),采用免疫荧光、体外细胞划痕实验观察各组细胞缺氧后(1%O2)其细胞骨架的主要成分α-微管蛋白(α-tubulin)的变化情况以及细胞迁移能力的变化。结果①缺氧0.5 h,细胞中MAP4的表达会升高,随着缺氧时间延长(缺氧1、3 h),表达又迅速降低;②早期缺氧后各组细胞的微管动力学均发生变化,细胞骨架趋于松散解聚以利于迁移;③缺氧24 h后正常细胞组和阴性对照组的迁移能力较常氧24 h明显增强(P<0.05);常氧状态下,干扰MAP4组细胞的迁移能力比正常细胞组和阴性对照组细胞明显减弱(P<0.01),缺氧后细胞迁移能力未见明显增强。结论缺氧微环境调控了HaCaT细胞MAP4的表达,进而影响了细胞微管动力学的变化,增强了细胞的迁移能力。
Objective To observe the expression of microtubule-associated protein 4 (MAP4) in human immortalized keratinocyte (HaCaT) cell line and its effect on cell migration in hypoxic environment. Methods Western blot was used to detect the expression of MAP4 in HaCaT cells after 1h hypoxia (1% O2, hypoxia time 0.5, 1, 3 h respectively); normal cell group was established to interfere MAP4 group and negative control group The changes of α-tubulin, the major component of cytoskeleton and the change of cell migration ability were observed by immunofluorescence and in vitro cell scratch assay after hypoxia (1% O2) . Results ① After 0.5 h hypoxia, the expression of MAP4 in the cells increased. With the prolonged hypoxia time (1 h and 3 h), the expression of MAP4 decreased rapidly. (2) The microtubule dynamics (P <0.05). In normal normoxic condition, the migration of MAP4 was inhibited in normal cells and negative control groups after 24 h hypoxia Migration of group cells was significantly weaker than that of normal cells and negative control group (P <0.01), and cell migration ability was not significantly increased after hypoxia. Conclusion Hypoxic microenvironment controls the expression of MAP4 in HaCaT cells, which in turn affects the dynamics of microtubules and enhances the migration of cells.