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目的:研究Hedgehog信号通路阻断剂(环巴胺)对DU145细胞增殖的抑制作用。方法:不同浓度(1、10、50、100μmol/L)环巴胺干预DU145细胞,分别在24、48、72h后采用噻唑蓝比色法检测其对细胞增殖的抑制作用;流式细胞术检测环巴胺对细胞周期的影响;RT-PCR检测50μmol/L环巴胺作用48h后实验组和对照组细胞周期蛋白E(cyclinE)mRNA表达水平的差异。结果:环巴胺对DU145细胞的抑制作用呈时效和量效依赖关系,当浓度>10μmol/L作用24h后会显著抑制细胞增殖,10、50、100μmol/L浓度组对细胞的抑制率分别为7.42%、12.70%和59.15%,与空白对照组相比,差异有统计学意义(P<0.05)。流式细胞术检测发现,当环巴胺浓度达10μmol/L以上,干预48h后G1期细胞比例明显升高。对照组、10、50μmol/L浓度组的G1期细胞百分比分别为:(52.17±2.21)%、(60.13±2.75)%和(74.30±3.52)%,差异有统计学意义(P<0.01);凋亡峰也随环巴胺浓度增加而逐渐增高。50μmol/L环巴胺作用48h后DU145细胞cyclinEmRNA表达显著降低,与空白对照组相比降低61.90%(P<0.01)。结论:环巴胺可以抑制DU145细胞的增殖能力,其机制可能与下调DU145细胞cyc-linEmRNA表达水平,从而将DU145细胞阻滞于G1期有关。环巴胺亦可以诱导DU145细胞凋亡。
Objective: To study the inhibitory effect of Hedgehog signaling pathway inhibitor (cyclopamine) on DU145 cell proliferation. METHODS: DU145 cells were treated with different concentrations of cyclopamine (1, 10, 50, and 100μmol / L), and the inhibitory effects on cell proliferation were detected by MTT assay 24, 48 and 72 hours later. Flow cytometry The effect of cyclopamine on cell cycle was analyzed by RT-PCR. The difference of cyclinE mRNA expression between experimental group and control group was detected by RT-PCR 48h after treatment with 50μmol / L cyclopamine. RESULTS: The inhibitory effect of cyclopamine on DU145 cells was time-dependent and dose-dependent. When the concentration of 10μmol / L was applied for 24 h, the cell proliferation was significantly inhibited. The inhibitory rates of 10, 50 and 100 μmol / L were 7.42%, 12.70% and 59.15%, respectively. The difference was statistically significant (P <0.05) compared with the blank control group. Flow cytometry showed that when the concentration of cyclopamine reached more than 10μmol / L, the proportion of cells in G1 phase increased significantly after 48h intervention. In control group, the percentage of cells in G1 phase was (52.17 ± 2.21)%, (60.13 ± 2.75)% and (74.30 ± 3.52)% in 10 and 50μmol / L groups, respectively. The difference was statistically significant (P <0.01) The apoptotic peak also increased gradually with the increase of cyclopamine concentration. The expression of cyclinEmRNA in DU145 cells was significantly decreased after treated with 50μmol / L cyclopamine for 48h, which was decreased by 61.90% (P <0.01) compared with the blank control group. CONCLUSION: Cyclopamine can inhibit the proliferation of DU145 cells. The mechanism may be related to the down-regulation of cyclin-1 mRNA expression in DU145 cells and thus arresting of DU145 cells in G1 phase. Cyclopamine can also induce DU145 cell apoptosis.