铜基配合物负载 VEGF-siRNA 协同增强肿瘤血管生成抑制

According to the reports, tumors can derive endothelial cells to generate their own vasculatures for providing the nutritions, promoting tumorigenesis, invasion, deterioration, and metastasis; and tumor cells are the roots resulting in overexpressed vascular endothelial growth factor (VEGF) which promote angiogenesis in tumors. Inhibiting angiogenesis and simultaneously killing tumor cells can achieve significant anti-tumor effects. Small interfering RNA has been the most promising nucleic acid for gene therapy in the field of human diseases. Many researches of various gene carriers improved the efficacy of siRNA treatment. However, such a gene carrier having anti-angiogenesis and anti-tumor functions has not been reported. Studies have shown that some copper-based complexes can inhibit angiogenesis and induce apoptosis of tumor cells. In this study we will synthesize the copper-based complexes which not only they can serve as the gene carriers, but also they can exhibit anti-angiogenesis and anti-cancer functions, loading VEGF-siRNA into target cells to release and silence the expression of VEGF gene, and then the chemotherapy of carriers and silencing efficacy of gene synergistically enhance anti-angiogenesis and anti-tumor functions. The xenografts nude mice are used to investigate their anti-angiogenesis and anti-cancer effects in vivo, and gel electrophoresis, flow cytometry, and laser scanning confocal microscope are used to investigate the cellular uptake and localization for elucidating the loading and releasing of siRNA from various angles, and tube formations and aortic ring microtubule formations assays are used to investigate the inhibition of angiogenesis, and western blot assays are used to investigate the expression of the key proteins involving angiogenesis in the downstreams of the VEGFR2 signaling pathway. This study provides the theoretical basis for copper-based complexes as siRNA carriers synergistically enhancing anti-tumor efficacy.

据报道，肿瘤可衍生内皮细胞形成自身的脉管系统提供营养、促进肿瘤发展浸润恶化和转移，而肿瘤细胞是血管生长因子VEGF过度表达的根源，促进肿瘤血管生长。抑制肿瘤血管生成同时杀死肿瘤细胞才能达到显著的抗肿瘤效果。小干涉RNA在基因水平上治疗疾病有远大前景。多类基因载体提高了siRNA治疗效果，但自身有抗血管生成和抗肿瘤功能的载体未见报道。研究表明，一些铜配合物能抑制血管生成和诱导肿瘤细胞凋亡。本研究合成既有载体功能又能抗肿瘤的铜配合物，负载VEGF-siRNA 进入靶细胞释放，沉默VEGF基因的表达，载体化学治疗与基因沉默效应协同增强抗血管生成抗肿瘤功能。裸鼠异种移殖研究体内抗血管抗肿瘤；电泳、激光共聚焦和流式细胞术研究细胞吸收及定位，阐明siRNA的负载与释放；管形成、动脉环出芽研究血管生成抑制；Western blot分析关键蛋白的调控。研究为铜配合物作为基因载体协同增强抗肿瘤提供理论依据。

据报道，肿瘤可衍生内皮细胞形成自身的脉管系统提供营养、促进肿瘤发展浸润恶化和转移，而肿瘤细胞是血管生长因子VEGF过度表达的根源，促进肿瘤血管生长。抑制肿瘤血管生成同时杀死肿瘤细胞才能达到显著的抗肿瘤效果。小干涉RNA在基因水平上治疗疾病有远大前景。多类基因载体提高了siRNA治疗效果，但自身有抗血管生成和抗肿瘤功能的载体未见报道。研究表明，一些铜配合物能抑制血管生成和诱导肿瘤细胞凋亡。本研究合成了多个既有载体功能又能抗肿瘤活性的铜配合物，它们负载VEGF-siRNA进入靶细胞释放，沉默VEGF基因的表达，实现了载体化学治疗与siRNA 基因沉默效应协同增强抗血管生成和抗肿瘤的功能。此外，本研究还合成了多个非基因载体的铜基活性配合物，研究结果表明这些配合物可以作为肿瘤血管抑制剂和有效地抗肿瘤作用。本研究的内容有：铜配合物的合成与结构表征；铜配合物纳米粒子的制备与表征；电泳、流式细胞术和电感耦合等离子体技术研究细胞吸收，激光共聚焦显微镜考察了细胞吸收及定位，阐明siRNA的负载与释放；流式细胞术分析诱导凋亡与损伤、检测细胞内活性氧水平以及线粒体膜电位坍塌；管形成、动脉环出芽研究血管生成抑制；裸鼠异种移殖以及肿瘤组织免疫组化或免疫荧光分析，探究体内抗血管生成与抗肿瘤的作用和机制； Western blot分析关键蛋白的调控。本研究为铜配合物作为基因载体协同siRNA增强抗肿瘤作用提供理论依据。