目的研究大黄酸衍生物大黄酸鬼臼毒素酯(RH-01)对人骨肉瘤(HOS)细胞的作用及其机制。方法体外培养HOS细胞,应用MTT法测定RH-01作用72h的IC₅₀值,羟基磷灰石吸附实验测定RH-01在体外的骨亲和性,荧光染色观察RH-01对HOS细胞形态的影响,流式细胞仪检测细胞周期分布情况,RT-PCR检测HOS细胞周期蛋白Cyclin D1,CDK4基因表达的变化。结果RH-01能明显抑制HOS细胞的生长,作用72h的IC₅₀值为0.18μmol/L;羟基磷灰石对四环素和RH-01吸附值分别为(8.1±0.15)、(14.8±0.11)μmol/g,RH-01的骨亲和性高于四环素;荧光染色后,可见到经RH-01处理的HOS细胞膜皱褶、卷曲、破裂,核碎裂固缩,聚集成细小的凝聚块;流式细胞仪检测到细胞周期G₂+M期阻滞明显;RT-PCR的半定量检测结果表明CyclinD1和CDK4 mRNA表达降低。结论RH-01通过调控细胞周期驱动机制使细胞发生周期阻滞,导致HOS细胞的生长抑制,并且RH-01在体外具有良好的骨亲和性。

Objective To study the effects of Podophyllotoxin rheinate (RH-01) on human osteoma sarcomatosum (HOS) cells. Methods HOS Cells were cultured in MEM alone or exposed to different concentrations of RH-01. The growth inhibition was analyzed by MTT assay. The bone-targeted effect of RH-01 was evaluated by values of hydroxyapatite ceramic adsorption. The flow cytometry (FCM), fluorescence microscopy, and RT-PCR assay were employed to detect the action mechanism of RH-01. Results RH-01 Remarkably inhibited HOS cells growth, its IC₅₀ value was 0.18 μmol/L at 72 h. Values of hydroxyapatite ceramic adsorption towards AcM and RH-01 were (8.1±0.15) and (14.8±0.11) μmol/g, respectively, which demonstrated that RH-01 had much stronger bone-targeted function than AcM. After the HOS cells with RH-01 diluted by Hoechst 33342 solution, it showed typically morphologic change compared with the control group under fluorescence microscope. Results indicated that RH-01 dramatically induced an accumulation in G₂/M phase of cell cycle at 24 h by FCM analysis. RT-PCR Assay showed that the expression of cyclin D1, CDK₄ mRNA obviously decreased in a concentration dependent manner. Conclusion The compound RH-01 could obviously inhibit the growth of HOS cell line and have a very strong bone-afinity feature in vitro.

国家自然科学基金资助项目(30371682)