Nocodazole对骨肉瘤MG-63细胞增殖凋亡的影响及机制研究

The role of Nocodazole in apoptosis of osteosarcoma MG-63 cells and its mechanism

目的以细胞有丝分裂阻滞剂Nocodazole为诱导物,诱导骨肉瘤MG-63细胞凋亡,并进行生物化学指标的检测。方法首先,观察1nmol/L～10μmol/L递增浓度的Nocodazole对MG-63细胞的作用,然后选取10,50,100,500nmol/L4个浓度诱导凋亡,通过噻唑蓝(MTT)比色实验测定细胞生长曲线;流式细胞技术(FCM)分析细胞周期;免疫细胞化学方法检测PLK1表达情况;用反转录-聚合酶链反应(RT-PCR)分析Nocodazole作用前后MG-63细胞内PLK1基因表达情况。结果不同浓度Nocodazole作用MG-63细胞24h后,对细胞增殖的抑制作用呈浓度依赖性增加。流式细胞仪分析结果显示,Nocodazole作用MG-63细胞后,G0/G1期细胞含量降低,S期细胞比例增加,G2/M期细胞比例则明显增加,且随着处理浓度的增加其G2/M期细胞含量逐渐增加,与对照组比较差异有统计学意义(P<0.05)。Nocodazole作用后,免疫细胞化学检测显示,PLK1表达明显下降。RT-PCR分析显示经Nocodazole作用后的骨肉瘤细胞株MG-63表达PLK1较用药前明显下降。上述结果都有明显的量-效关系。结论 Nocodazole诱导MG-63细胞凋亡、G2/M期阻滞的机制可能是通过抑制PLK1基因的表达实现的。

Objective To induce the apoptosis of osteosarcoma MG-63 cells using cell mitosis inhibitor Nocodazole as an inducer and to study its biochemical features.Methods Firstly,the effects of Nocodazole at gradient concentrations between 1 nmol/L^10 μmol/L on MG-63 cells were observed;then the apoptosis of MG-63 cells was induced by using four concentrations of Nocodazole（10,50,100,500 nmol/L）,respectively,and MTT colorimetry was used to generate the growth curve of MG-63 cells.Before and after Nocodazole induction,the cell cycle was analyzed by flow cytometry（FCM）,expression of PLK1 detected by immunocytochemistry,and expression of PLK1 of osteosarcoma cells line MG-63 analyzed by RT-PCR.Results Nocodazole showed an increasing inhibitory effect on MG-63 cell proliferation in a dose-dependent manner 24-hour induction with different concentrations.Outcomes of flow cytometry revealed lowered percentage of G0/G1 phase cells and significant increase in the proportion of both S-phase and G2/M phase cells.Notably,the number of G2/M phase cells increased along with the increasing concentration,which was statistically different from that in the control group（P0.05）.After Nocodazole induction,immunocytochemistry showed a remarkable decrease of PLK1 expression,and RT-PCR displayed obviously down-regulated expression of PLK1 mRNA of osteosarcoma cells line MG-63 as compared with before medication.Conclusion The mechanism of Nocodazole-induced apoptosis in MG-63 cells and G2/M phase arrest may be achieved by inhibited PLK1 gene expression.