去甲斑蝥素对胆管癌RBE细胞α-Tubulin骨架蛋白及细胞周期的影响

Norcantharidin induces G2/M cell cycle arrest though disrupting α-Tubulin cytoskeleton

目的观察去甲斑蝥素对人胆管癌RBE细胞周期的影响及对微管蛋白(α-Tubulin)的作用。方法体外培养胆管癌RBE细胞,分别用不同剂量去甲斑蝥素处理,流式细胞学检测细胞周期的影响,免疫组化染色法观察α-Tubulin结构变化,Real-time PCR检测α-Tubulin mRNA转录水平变化。结果流式细胞学检测显示胆管癌RBE细胞随着药物浓度的增加,G2/M期阻滞作用增强,10、20、40μg/ml组与对照组相比具有统计学意义(P=0.000,P<0.01)。免疫组化染色法观察到去甲斑蝥素作用下,胆管癌RBE细胞α-Tubulin骨架结构被破坏,Real-time PCR证实去甲斑蝥素使胆管癌RBE细胞α-Tubulin转录水平减少,差异具有统计学差异(P=0.002,P=0.000,P<0.01)。结论去甲斑蝥素可以通过破坏胆管癌RBE细胞Tubulin骨架、抑制α-Tubulin mRNA的表达,阻止纺锤体的形成,将细胞周期阻滞在G2/M期,导致生长抑制,是其抗肿瘤机制之一。

Objective To explore the mechanisms of Norcantharidin＇s anti-tumor effect by investigating cell cycle and α-Tubulin cytoskeleton changes in cholangiocarcinoma cell line RBE after treated by Norcantharidin. Methods The RBE cells were cultured in vitro, followed by treating with various concentration of norcantharidin for 24 h. Cell cycle changes were detected by flow cytometric analysis. We used immunohistochemical （IHC） to observe α-Tubulin cytoskeleton changes, and real-time PCR was used to detect α-Tubulin mRNA transcript levels. Results The result of flow cytometric analysis shown the cell cycle arrested at G2/M. The damages of α-Tubulin cytoskeleton under the treatment of Norcantharidin were observed by IHC, and real-time PCR determined that α-Tubulin was decreased at mRNA level. Conclusions Norcantharidin can prevent the formation of spindle to arrest the cell cycle in G2/M phase, by disrupting Tubulin cytoskeleton, inhibiting α-Tubulin mRNA expression, which is one of the mechanisms of Norcantharidin＇s anti-tumor effect.