金属富勒醇Gd@C82(OH)22调控血管内皮生长因子受体（VEGFR）跨膜信号转导的机制研究

Metallofullerene Gd@C82 (OH)22 nanoparticles have been demonstrated to successfully inhibit the tumor growth and metastasis via modulating tumor microenvironment. In our preliminary studies, Gd@C82 (OH)22 nanoparticles exhibited an obvious anti-angiogenesis potency. However, the molecular mechanism underlying the anti-angiogenesis induced by Gd@C82 (OH)22 nanoparticles has not been fully elucidated yet. In the present study, based on the structural characteristics and the physicochemical property of Gd@C82 (OH)22 nanoparticles , we will explore the interaction between Gd@C82 (OH)22 nanoparticles and vascular endothelial growth factor(VEGF) and its receptor VEGFR and the subsequent impact on VEGFR-mediated transmembrane signaling pathways by a combinatorial application of physical, chemical and biological methods. The binding sites number and the apparent binding constant of Gd@C82 (OH)22 nanoparticles and VEGF, VEGFR will be measured by UV-Vis absorption and fluorescence spectroscopy. Furthermore, the conformation change of VEGF, VEGFR upon the binding of Gd@C82 (OH)22 nanoparticles as well as the intervention of the VEGF-VEGFR binding by the nanoparticles will be detected by small angle X-ray scattering(SAXS) and synchrotron radiation circular dichroism (SRCD). Finally, molecular dynamics simulations will be used to address the intrinsic recognition and binding property to VEGF or VEGFR of Gd@C82 (OH)22 and its potential inhibition effect on VEGF-VEGFR binding. All of these will help us to elucidate how Gd@C82 (OH)22 nanoparticles modulate VEGFR-mediated transmembrane signaling involved in its anti-angiogenesis effect , to define the possible structure-activity relationship of nanomedicine, which will be of great significance to promote the possible medical applications of other nanoparticles bearing the potential therapeutic effects in the future.

Gd@C82(OH)22纳米颗粒被证实能通过调控肿瘤微环境高效抑制肿瘤的生长与转移。前期研究发现它能显著抑制肿瘤组织新生血管的形成，但其分子机制有待深入研究。本研究拟从Gd@C82(OH)22的结构特点入手，研究它与VEGF和VEGFR的相互作用及其对VEGFR介导的跨膜信号转导的影响。拟用紫外可见吸收光谱和荧光光谱测定Gd@C82(OH)22与VEGF、VEGFR的结合常数及结合个数，用同步辐射圆二色谱和小角散射技术检测Gd@C82(OH)22对VEGF、VEGFR的分子构象及结合的影响，最后用分子动力学模拟技术研究Gd@C82(OH)22与VEGF,VEGFR的结合过程和结合位点，确定Gd@C82(OH)22调控VEGFR介导的跨膜信号转导的方式，初步探索Gd@C82(OH)22的结构及性质与其抗肿瘤效应之间的关系，这对促进其他类似的具有潜在治疗作用的纳米颗粒在医学上的应用具有重要意义

癌症是人类健康的主要威胁。快速发展的纳米技术有望给现有癌症诊疗带来革命性突破。金属富勒醇Gd@C82(OH)22被证实能通过调控肿瘤微环境高效抑制肿瘤生长与转移。在被发现的诸多抗癌药理机制中，Gd@C82(OH)22的抗肿瘤作用与铁代谢的研究未被涉及。但越来越多的研究证据表明铁代谢与肿瘤微环境密切相关。肿瘤细胞对微环境中铁的摄入主要通过转铁蛋白完成。当Gd@C82(OH)22经静脉注射进入血液后，由于其表面的高反应性，很有可能与血浆中高丰度的转铁蛋白相互作用。因此Gd@C82(OH)22的抗肿瘤作用可能与铁转运相关。本研究（课题计划在原计划基础上调整，原因在正文中说明）从Gd@C82(OH)22结构特点入手，通过研究其与转铁蛋白的相互作用，阐明其影响铁转运的机理及其抗癌机理。同时以C60(OH)22为对照结构，旨在探索纳米物质的结构-生物学效应的关系。用外加铁和转铁蛋白的方式来模拟高水平铁和转铁蛋白高表达的肿瘤微环境来研究其对肿瘤细胞生长的影响。结果发现外加铁或转铁蛋白对肿瘤细胞均有促增殖作用，Gd@C82(OH)22与C60(OH)22缓解外加铁或转铁蛋白的促肿瘤细胞增殖效应且与减少的铁摄入密切相关。利用小角散射技术研究Gd@C82(OH)22和C60(OH)22对转铁蛋白结构和形状的影响。结果发现转铁蛋白在溶液中以二聚体形式存在。当氯化铁加入到转铁蛋白溶液中，铁离子（Fe3+）与转铁蛋白上的铁结合位点结合，使转铁蛋白二聚体变成单体。当Gd@C82(OH)22加入到转铁蛋白溶液中，检测到转铁蛋白-Gd@C82(OH)22的体积约为两倍转铁蛋白二聚体的体积且形状规则，推测有结构对称的转铁蛋白四聚体形成。转铁蛋白四聚体的形成使转铁蛋白受体对其识别失败。在C60(OH)22-转铁蛋白混合体系中，转铁蛋白还是单体且C60(OH)22-转铁蛋白的形状与铁-转铁蛋白形状差异较大。Gd@C82(OH)22和C60(OH)22对另一种高丰度血浆蛋白IgG构象无影响，说明Gd@C82(OH)22对转铁蛋白的作用有特异性。由此从结构角度提供了Gd@C82(OH)22和C60(OH)22抑制肿瘤细胞铁摄入的科学性解释，这是首次提供的基于实验非理论模拟获取的Gd@C82(OH)22与蛋白相互作用的信息，也说明通过纳米结构的智能设计和开发调控纳米物质生物学效应的可行性。