雷帕霉素抑制人骨肉瘤MG-63细胞周期及CyclinD1表达实验研究.pdf

摘 要 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：体外培养人骨肉瘤MG-63 细胞，用不同浓度的雷帕霉素(1nmol/L、10nmol/L、 25nmol/L)干预MG-63 细胞。采用CCK-8 试剂盒，运用水溶性四唑盐WST-8 比色法 检测各实验组MG-63 细胞增殖的变化，计算细胞增殖抑制率；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： CCK-8 检测结果显示雷帕霉素能显著抑制MG-63 细胞增殖，呈时间依赖性， 药物作用时间越长，细胞增殖抑制率越高，差别有显著性意义(P ＜0.05) ，但各浓度 雷帕霉素组间细胞增殖抑制率差异无显著性意义(P ＞0.05) ；流式细胞分析结果显示 雷帕霉素能显著抑制细胞周期，各浓度雷帕霉素组与对照组相比G0/G1 期细胞明显增 多，S 期细胞明显减少，差别有显著性意义(P ＜0.05) ；RT-PCR 检测显示各浓度雷帕 霉素组与对照组相比MG-63 细胞Cyclin D1 mRNA 的表达差异无显著性意义 (P ＞ 0.05) ，雷帕霉素对MG-63 细胞Cyclin D1 mRNA 表达无明显影响；免疫细胞化学分 析结果显示雷帕霉素能显著抑制Cyclin D1 蛋白的表达，呈浓度依赖性，差别有显著 性意义(P ＜0.05) 。 结论：雷帕霉素能显著抑制MG-63 细胞增殖及细胞周期，下调MG-63 细胞Cyclin D1 蛋白的表达，但对MG-63 细胞Cyclin D1 mRNA 的表达无明显影响。 关键词：雷帕霉素；骨肉瘤；细胞周期；细胞周期蛋白D1 Ⅰ Abstract Objective：To investigate the inhibitive effects of cell cycle and Cyclin D1 expression by rapamycin in MG-63 cells and probe the possible mechanism. Method ：MG-63 cells were cultured in vitro and treated with different concentration of rapamycin(1nmol/L 、10nmol/L、25nmol/L). Used CCK-8 by water-soluble tetrazolium salt(WST-8) assay to detect the growth inhibitive effects in MG-63 cells treated with rapamycin ；Flow cytometry was used to analysis MG-63 cell cycle ；The gene expression of Cyclin D1 was examined by RT-PCR and immunocytochemistry SABC. Result ：CCK-8 assay showed that the growth of MG-63 cells was significantly inhibited by rapamycin, it revealed a time-dependent relationship (P ＜0.05).The longer the rapamycin treated, the higher the cell proliferation inhibition rate performed. But there was no significantly different between three rapamycin groups(P ＞0.05) ；Flo