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目的使用四环素操纵子(Tet-on)可控表达系统研究细胞周期蛋白依赖性激酶抑制蛋白2A(CDKN2A/p16~(INK4a))对MCF7乳腺癌细胞形态及细胞骨架的影响。方法以p16~(INK4a)表达缺失的MCF7乳腺癌细胞为宿主细胞,用慢病毒通过“两步感染法”建立MCF7-p Tet-on-p16~(INK4a)可诱导表达细胞系,加入诱导剂强力霉素(Dox)诱导目的基因表达;细胞计数检测细胞生长变化;采用免疫细胞化学染色检测上皮钙黏素(E-cadherin)表达,采用鬼笔环肽(phalloidin)标记纤维型肌动蛋白(F-actin),观察p16~(INK4a)对细胞间黏附连接、细胞骨架的影响;用显微镜对细胞拍照后,用Image J软件进行形态分析。结果成功建立了可诱导表达p16~(INK4a)的MCF7稳定细胞系,Dox可以有效诱导p16~(INK4a)的表达;诱导条件下稳定表达p16~(INK4a)的MCF7细胞增殖受抑制,细胞体积增大,轮廓线延长;而对照MCF7细胞F-actin主要定位于胞质,p16~(INK4a)表达的细胞内F-actin向细胞边缘分布增加。结论 p16~(INK4a)在MCF7细胞中的表达可以导致细胞体积增大、细胞间黏附连接弱化、F-actin向细胞外周重新分布。
Objective To investigate the effects of cyclin-dependent kinase inhibitor 2A (CDKN2A / p16 INK4a) on the morphology and cytoskeleton of MCF7 breast cancer cells using Tet-on controllable expression system. Methods MCF7 breast cancer cells with p16 INK4a expression were used as host cells and MCF7-p Tet-on-p16 INK4a cells were induced by lentivirus using the two-step infection method. The expression of E-cadherin was detected by immunocytochemical staining, and the fibroid motility was detected by phalloidin (F-actin) was used to observe the effect of p16 INK4a on cell adhesion and cytoskeleton. The cells were photographed by microscope and analyzed by Image J software. Results The stable cell line MCF7 that could induce the expression of p16 INK4a was successfully established. Dox could effectively induce the expression of p16 INK4a. The proliferation of MCF7 cells stably expressing p16 INK4a was inhibited and the cell volume increased While the control MCF7 cells F-actin mainly located in the cytoplasm, p16 INK4a expression of intracellular F-actin to the cell edge distribution increased. Conclusion The expression of p16 INK4a in MCF7 cells can lead to the increase of cell volume, the weakening of intercellular adhesion and the redistribution of F-actin to the periphery of cells.