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目的观察不同浓度有丝分裂阻滞剂Nocodazole对Hela细胞株G_2/M期同步化的调节作用及其对纺锤体结构的影响。方法分别以3.0、1.0、0.3μmol/L Nocodazole处理Hela细胞(Nocodazole 3.0、1.0、0.3μmol/L处理组)18 h。于Nocodazole撤除后0(处理18 h)、3、6、9 h时间点收集细胞,流式细胞仪检测G_2/M期细胞百分比;间接免疫荧光染色激光共聚焦显微镜观察细胞有丝分裂器纺锤体α-微管蛋白(α-tubulin)排列。以不添加Nocodazole处理的Hela细胞作为对照组。结果Nocodazole处理18 h,Nocodazole 3.0、1.0、0.3μmol/L处理组Hela细胞G_2/M期细胞百分比分别为55.95%、51.09%和47.81%,均显著高于对照组的9.54%(P<0.05)。在Nocodazole撤除后3、6、9 h时间点,Nocodazole 3.0μmol/L和1.0μmol/L处理组G_2/M期细胞百分比无明显变化;而Nocodazole 0.3μmol/L处理组G_2/M期细胞百分比下降明显(30.43%、12.91%、10.23%),撤除后6 h时间点的G_2/M期细胞百分比与对照组比较差异已无统计学意义(P>0.05)。α-tubulin荧光染色激光共聚焦显微镜观察显示,Nocodazole撤除后Nocodazole 0.3μmol/L处理组G_2/M期细胞微管迅速再聚合形成两极纺锤体且纺锤丝结构清晰。结论Nocodazole对Hela细胞G_2/M期同步化具有调节作用;药物撤离后,0.3μmol/L Nocodazole处理组细胞周期恢复迅速且对纺锤体结构影响最小。
Objective To observe the effects of different concentrations of mitotic blocker Nocodazole on G 2 / M phase synchronization in Hela cells and its effect on spindle structure. Methods Hela cells (Nocodazole 3.0, 1.0 and 0.3 μmol / L) were treated with 3.0, 1.0 and 0.3 μmol / L Nocodazole for 18 h. The cells were harvested at 0 (18 h) and 3, 6, and 9 h after Nocodazole removal. The percentage of cells in G 2 / M phase was detected by flow cytometry. The expression of spindle mitochondrial α- Tubulin arrangement. Hela cells without Nocodazole treatment served as control group. Results After Nocodazole was treated with Nocodazole for 18 h, the percentages of cells in G 2 / M phase of Hela cells treated with 3.0, 3.0 and 3.0 μmol / L Nocodazole were 55.95%, 51.09% and 47.81%, respectively, which were significantly higher than those of control group (9.54%, P 0.05) . At 3, 6, and 9 h after Nocodazole removal, there was no significant change in the percentage of cells in G 2 / M phase of Nocodazole 3.0 μmol / L and 1.0 μmol / L groups, while the percentage of cells in G 2 / M phase of Nocodazole 0.3 μmol / L decreased Significantly (30.43%, 12.91%, 10.23%), the percentage of cells in G 2 / M phase at 6 h after withdrawal had no significant difference compared with the control group (P> 0.05). α-tubulin fluorescence staining laser confocal microscopy showed that Nocodazole removed Nocodazole 0.3μmol / L treatment group G_2 / M phase microtubules quickly re-polymerization to form bipolar spindle spindle structure and clear. Conclusion Nocodazole regulates G 2 / M phase synchronization in Hela cells. After drug withdrawal, the cell cycle recovered rapidly with 0.3 μmol / L Nocodazole treatment and had the least effect on the spindle structure.