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目的观察hsMAD2过表达以及联合Nocodazole处理对HepG2细胞的生长和细胞周期的影响。方法将人全长hs-MAD2基因经脂质体包裹转染肝细胞肝癌HepG2细胞,经G418筛选期获得转基因癌细胞克隆,采用免疫细胞化学法、Westernblot方法检测hsMAD2的表达情况。观察细胞形态、测量生长曲线和分裂指数曲线,观察hsMAD2基因对HepG2细胞生长和细胞增殖能力的影响,流式细胞仪检测HepG2细胞周期的变化。然后观察在纺锤体阻滞剂50nmol/L Nocodazole作用下,hs-MAD2对HepG2细胞的影响。结果稳定表达外源性hsMAD2蛋白的HepG2细胞生长速度和细胞增殖能力并无变化;流式细胞仪检测结果显示HepG2-hsMAD2细胞周期G2、S期比例增加,而G1期细胞比例下降,但差异无显著性。经过nocodazole处理后,HepG2-hsMAD2细胞生长速度下降,差异具有显著性,细胞周期出现G2期阻滞。相反,HepG2在受到Nocodazole处理后无明显变化。结论单纯过表达外源性hsMAD2对细胞无影响。由于hsMAD2蛋白发挥了细胞周期关卡作用,使受到No-codazole破坏作用的细胞出现细胞周期的阻滞。
Objective To observe the effect of hsMAD2 overexpression and Nocodazole treatment on the growth and cell cycle of HepG2 cells. Methods Human full-length hs-MAD2 gene was transfected into HepG2 hepatocellular carcinoma cells by lipofectamine. The clones of transgenic cancer cells were obtained by G418 screening. The expression of hsMAD2 was detected by immunocytochemistry and Western blot. The morphological changes of HepG2 cells were observed. The growth and proliferation of HepG2 cells were observed by flow cytometry. The cell cycle of HepG2 cells was detected by flow cytometry. Then observe the effect of hs-MAD2 on HepG2 cells under the action of spindle inhibitor 50nmol / L Nocodazole. Results The growth of HepG2 cells stably expressing exogenous hsMAD2 protein did not change. The results of flow cytometry showed that the proportion of cells in G2 phase and S phase of HepG2-hsMAD2 cells increased while the proportion of cells in G1 phase decreased but the difference was not Significance. After nocodazole treatment, HepG2-hsMAD2 cell growth rate decreased, the difference was significant, G2 cell cycle arrest. In contrast, HepG2 showed no significant change after Nocodazole treatment. Conclusion Overexpression of exogenous hsMAD2 has no effect on the cells. As the hsMAD2 protein exerts a cell cycle checkpoint, cell cycle arrest occurs in cells that are damaged by No-codazole.