【作者】 李娜；

【导师】 吴建华；

【作者基本信息】 华南理工大学 ， 生理学， 2016， 硕士

【摘要】 目前,恶性肿瘤的发病人数和其导致的死亡人数每年都在急剧上升,已经成为低收入和中等收入家庭沉重的负担。因此,寻找有效诊断和治疗恶性肿瘤的方法成为迫切的任务,关于肿瘤的发生发展过程的研究也成为了当今的研究热点。整合素通过介导细胞与细胞之间、细胞与细胞外基质之间的相互作用,在恶性肿瘤的发生和肿瘤细胞的侵袭转移、伤口愈合以及炎症等病理过程中扮演着非常重要的角色。其中,α_vβ₃是整合素家族中最受关注的成员之一。研究发现,α_vβ₃在多种恶性肿瘤细胞的表面以及新生血管内皮细胞中表达水平较高,在肿瘤血管的生成过程中起到非常关键的作用。因此,整合素α_vβ₃已经成为肿瘤诊断和肿瘤治疗的重要靶点之一。鉴于含RGD序列短肽能够与整合素α_vβ₃快速且特异性结合,经放射性核素标记的含RGD序列短肽作为显影剂或药物载体等,已经广泛应用在肿瘤诊断和肿瘤治疗等相关医学领域。目前,含RGD序列短肽的设计主要有环形、线性两种不同构型,其与整合素α_vβ₃受体的亲和力不同,能够直接影响到抗肿瘤药物的治疗效果。为了揭示含RGD序列配体的构型如何影响其与α_vβ₃受体之间相互作用的机制,本研究利用分子动力学模拟建立环形c（RGDfV）和线性RGDfV各自与整合素α_vβ₃之间相互作用的实验模型,分析两种不同构型的含RGD序列配体与α_vβ₃之间相互作用的结构基础。结果显示,由于线性含RGD序列短肽自身结构比较灵活性,不仅容易与受体周围的残基发生相互作用,而且能形成分子内氢键,致使其与整合素α_vβ₃的结合稳定性明显比环形含RGD配体弱。本研究还发现,相比线性含RGD序列短肽,环形含RGD配体与受体相互作用中的Asp^RGD-MIDAS的静电能要强得多。这也是导致环形含RGD序列短肽与整合素α_vβ₃的结合更稳定的原因之一。在血流流体环境中,整合素α_vβ₃与含RGD序列短肽之间的相互作用是一个力与化学偶联的生物学过程,该过程不仅取决于其分子的化学本质,血流剪应力也起到重要的作用。本研究利用拉伸分子动力学模拟方法,在原子水平上研究拉力是如何调控α_vβ₃受体与含RGD序列配体之间的相互作用。研究发现,在整合素α_vβ₃与含RGD序列配体之间的解离过程中,主要有两个峰值,对应着两对关键的残基,分别是Arg^RGD-Asp²¹⁸和Asp^RGD-MIDAS;相比线性含RGD序列短肽,环形含RGD序列短肽与α_vβ₃的解离发生时断裂力较大,对应的断裂时间也相对较长,表明两者间的结合亲和力更高。而在整合素α_vβ₃与含RGD序列配体之间的结合过程中,相比线性RGD配体,环形RGD配体与整合素受体之间的相互作用能更强,两者间相互作用的关键残基对Asp^RGD-MIDAS也能保持在较近的作用距离内。以上实验说明,环形含RGD序列短肽与整合素α_vβ₃的结合更稳定。相对线形RGD配体,环状构型的含RGD序列短肽更有利于其与整合素α_vβ₃的结合。总之,本研究主要对整合素α_vβ₃与不同构型的含RGD序列短肽之间的相互作用的结构基础以及动力学机制进行研究,在原子层面上揭示含RGD序列短肽的构型如何影响其与整合素α_vβ₃的结合的稳定性以及亲和能力,证实了环化有利于提高配体与整合素α_vβ₃之间相互作用的稳定性和结合强度。研究结果有利于更深层次理解含RGD序列短肽的构型对其靶向识别整合素α_vβ₃的影响,为新型抗肿瘤药物的设计和开发提供有效的指导信息。更多还原

【Abstract】 At present,since the incidence of malignant tumor and its mortality is gradually increased every year,it has become a heavy burden in low-and middle-income families.Therefore,it is an urgent task to find out the ways to effectively treat malignant tumor,and study about the development of malignant tumor has become the research focus.Integrin plays an important role in some pathological process such as the invasion and metastasis of malignant tumor,wound healing and inflammation through mediated by cell and cell,and cells and extracellular matrix.Among integrin,α_vβ₃ is one of the important members of the integrin family.It’s reported that integrin α_vβ₃ is widely expressed on the surface of a variety of malignant tumor cells and vascular endothelial cells,and plays a critical roles in tumor angiogenesis.Consequently,integrin α_vβ₃ has become one of the important targets for tumor diagnosis and treatment.For the preferential and specific binding to integrin α_vβ₃,the RGD peptides labeled with radioactive nuclide were used as imaging or drug carrier which have been widely applied in the related medical field of imaging constructs and drug delivery.At present,the RGD-containing peptides are mainly designed into two different steric conformation: cyclic and linear.The affinity of RGD-containing peptides with integrin α_vβ₃ may be affected by steric conformation of the peptides that can directly affect the therapeutic effect of antitumor drug.To elucidate the mechanism of how the steric conformation of the RGD-containing peptides to affect the interaction between RGD peptides with integrin α_vβ₃,molecular dynamics simulation was used in this study to build the experiment models: the cyclic（RGDfV）and the linear RGDf V ligated with integrin α_vβ₃,respectively,and the structure mechanism of two different steric conformational RGD-containing ligands interact with α_vβ₃ was analyzed.The results showed that the binding stability of the linear RGD peptides ligated with integrin α_vβ₃ was much weaker than the cyclization,due to its flexibility that not only easy to interact with the surrounding residues around the receptor,but also can form intramolecular hydrogen bonds.Furthermore,it also found that compared with the linear RGD-containing peptides,the kinetic energy barrier between the Asp^RGD-MIDAS in the cyclization was much greater,which may be one of the reason why the cyclic RGD-containing peptides ligated with integrin α_vβ₃ are more stable.In the blood flow of fluid environment,the interaction between integrin α_vβ₃ and RGD-containing short peptides is a force-chemical coupling biological processes.It is not only regulated by the chemical properties of the molecular but also the blood flows.It.This study used steered molecular dynamics simulation to reveal how the tensile force to regulate the bonding strength between integrin α_vβ₃ and RGD-containing ligands at the atomic level.It was found that there were two major force peaks during the dissociation between integrin α_vβ₃ and RGD ligands,correspond to two important residues pairs,Arg^RGD-Asp²¹⁸ and Asp^RGD-MIDAS,respectively.Compared with that of the linear RGD-containing peptides,the rupture force was greater during the cyclic RGD peptide were pulling away from integrin α_vβ₃,while the disrupting time was longer.This suggests that the binding affinity between the cyclic RGD peptide and integrin α_vβ₃ are more potent.While in the process of the association between integrin α_vβ₃ and RGD-containing peptides,compared with the linear RGD ligands,the interaction energies between the cyclic RGD ligands with the receptor was much stronger,and the distance of the important residues pairs Asp^RGD-MIDAS was closer.The above experiment showed that the binding properties between the cyclic RGD peptide and integrin α_vβ₃ are more stable.Compared with the linear RGD ligands,the cyclization is employed to improve the binding properties with integrin α_vβ₃.In summary,this study was mainly focused on the structural and dynamic mechanism of integrin α_vβ₃ interacting with different steric conformation of RGD-containing peptides,and to explore how the steric conformation affect the stability and affinity between RGD-containing peptides with integrin α_vβ₃.It was confirmed that the cyclization could improve the binding stability and properties of RGD-containing peptides ligated with integrin α_vβ₃.The results of this study contribute to better understanding how the different steric conformation affecting the interaction between the RGD-containing sequence with integrin α_vβ₃,and offer effective guidance for the design and development of new antitumor drugs.更多还原