摘要
目的:揭示新型重组人内皮抑素(Endostar,恩度)与细胞毒药物顺铂联合使用的抗血管形成作用,探讨联合用药方式产生协同作用的分子机制。方法:在人脐静脉内皮细胞(HUVECs)培养体系上,通过增殖抑制分析、克隆形成分析、诱导凋亡、迁徙/侵袭分析以及管道形成研究,系统地考察恩度联合顺铂抑制血管形成的作用,并以鸡胚尿囊膜(CAM)模型观察和评价联合用药方式的抗血管形成特点。其后,以DNA微阵列技术展示不同药物干预HUVECs的早期基因表达谱,鉴定出重要的调节血管形成的效应基因,探讨这些基因在联合用药时发挥抗血管形成增效作用的分子机制。最后,以实时定量RT-PCR验证目标基因的表达。结果:恩度与顺铂联合使用在体外有协同抑制血管形成的作用,这种协同作用具有血管内皮细胞特异性。CAM分析表明恩度有显著的抗血管形成活性,其效应与剂量之间无线性关系,恩度与顺铂联合使用在体内也显示出协同的抗血管形成作用。通过基因表达谱研究,筛选出一些参与血管形成增效作用的重要基因,如JUNB、GATA2、INSR、F7和WNT1等。实时定量RT-PCR的验证结果与目标基因的芯片资料基本吻合。结论:恩度与顺铂联合使用具有协同的抗血管形成作用,其分子机制涉及到对上述重要基因表达的调节,这些基因可能通过VEGF/VEGFR信号途径、凝血级联以及WNT信号途径等参与抗血管形成的增效作用。
Objective: To reveal the synergistic antiangiogenic effects and mechanisms of novel recombinant human endostatin (Endostar) combined with cytotoxic drug cisplatin. Methods: On the cultivated system of human umbilical vein endothelial cells (HUVECs), we systematically investigated the antiangiogenic effects of Endostar combined with cisplatin, using the methods including proliferation inhibition assay, clonogenic assay , induction apoptosis, migration/invasion assay, and tube formation study, and in the chicken chorioallantoic membrane (CAM) assay, we observed and evaluated the antiangiogenic properties of combined modality. Meanwhile, we displayed the early gene expression profiles of different therapeutic modalities on HUVECs using DNA microarray technology, and identified some important genes involved in angiogenic modulation. We discussed the roles of these genes contributed to enhanced antiangiogenic effects, and confirmed the gene-chip data using real-time quantitative RT-PCR analysis. Results: There was a synergistic antiangiogenic effect in vitro when Endostar was used with cisplatin, and this synergistic effect was specific on endothelial cells. Endostar had notable antiangiogenic activity, with a nonliner dose-effect relationship, and combined Endostar with cisplatin exhibited a synergistic antiangiogenic effect in vivo. Some important genes associated with enhanced antiangiogenic action, such as JUNB, GATA2, INSR, F7, and WNT1, etc, were identified. The chip data of the target genes were mainly in accordance with the results of real-time quantitative RT-PCR. Conclusions: Combined Endostar with cisplatin resulted in a synergistic antiangiogenic effect, and the molecular mechanism was involved in the significant regulation of some genetic expression. These effectors could enhance the antiangiogenic effect through VEGF/VEGFR, coagulation cascade, and WNT signaling pathways.
引文
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