雷帕霉素抑制人骨肉瘤MG-63细胞周期的实验研究

Inhibitive effect of rapamycin on cell cycle in MG-63 human osteosarcoma cells

目的观察雷帕霉素对人骨肉瘤细胞株MG-63增殖、周期及Cyclin D1基因表达的影响,探讨雷帕霉素抑制骨肉瘤细胞周期的可能机制。方法体外培养骨肉瘤MG-63细胞,用不同浓度的雷帕霉素(1、10、25nmol/L)干预MG-63细胞。采用水溶性四唑盐WST-8比色法检测MG-63细胞增殖的变化;流式细胞仪检测MG-63细胞周期;RT-PCR及免疫细胞化学SABC法检测MG-63细胞Cyclin D1基因表达的变化。结果雷帕霉素能显著抑制MG-63细胞增殖,呈时间依赖性,差别有显著性意义(P<0.05);流式细胞分析显示雷帕霉素能显著抑制细胞周期,使G0/G1期细胞增多(P<0.05);RT-PCR检测显示雷帕霉素对MG-63细胞Cyclin D1mRNA表达无明显影响(P>0.05);免疫细胞化学分析雷帕霉素能显著抑制Cyclin D1蛋白的表达,差别有显著性意义(P<0.05)。结论雷帕霉素能下调MG-63细胞Cyclin D1蛋白的表达,抑制MG-63细胞增殖及细胞周期。

Objective To observe the effects of rapamycin on the multiplication and cycle of cell line MG-63 and gene expression of cyclin D1, and to investigate the possible mechanism for the inhibition of rapamycine on the cycle of osteosarcoma cells. Methods MG-63 cells were cultured in vitro and treated with rapamycine solution of different concentrations （lnmol/L, 10nmol/L, 25nmol/L）. Water-soluble tetrazolium salt（WST-8） assay was used to detect the change of multiplication of MG-63 cell. Flow cytometry was used to measure MG-63 cell cycle. RT-PCR and immunocytochemistry SABC were used to detect the gene expression of Cyclin D1. Results The growth of MG-63 cells was significantly inhibited by rapamycin. It revealed a time-dependent relationship （P〈0.05）. Flow cytometric analysis showed rapamycin significantly inhibited the cell cycle, and increased the number of cells in G0/G1 phase （P〈0.05）. RT-PCR test showed rapamycie had no significant effect on the expression of Cyclin D1mRNA （P〉0.05）. Immunocytochemistry analysis demonstrated rapamycine inhibited the expression of protein of Cyclin D 1 （P〈0.05）. Conclusions Rapamycin can down-regulate the expression of Cyclin D1 protein, and the multiplication and cell cycle of MG-63 cells.