Rap1GAP通过ERK和Akt途径抑制内皮细胞增殖、迁移的实验研究

发布时间：2018-05-10 11:31

本文选题：Rap1GAP + Rap1　；参考：《华中科技大学》2011年博士论文

【摘要】：研究目的 Rap1是小分子G蛋白Ras家族成员之一,体外的实验已经证明Rap1在细胞内发挥多种作用。Rap1对内皮细胞的作用主要是参与对内皮细胞间粘连形成、通透性等功能的调节。最近的研究表明,在内皮细胞中,Rap1不仅仅局限于细胞粘连,而且可以调节内皮细胞对一些促进血管新生刺激因素的反应。换句话说,Rap1在体内可以作为一种正性血管新生调节因素。 Rap1GAP是小分子G蛋白Rap1的GTP酶激活蛋白,它可以加速Rap1由与GTP结合的活化形式转变为与GDP结合的去活化形式的过程。一些研究者发现Rap1GAP可以失活Rap1,抑制Rap1介导的血管新生。但也有一些实验结果并不支持这一结论。因此,很有必要进一步研究Rap1GAP在血管新生的作用。揭示Rap1GAP在血管新生的作用有利于进一步了解血管新生的机理,并且能为与血管新生相关疾病提供一个新的、特异的研究切入点。血管新生是微血管从已经存在的血管床以出芽的方式长出,并逐渐形成新的血管分支及毛细血管丛的过程,它由一系列相互协调密切配合的过程来组成的,许多因素参与了对其的调节。PI3K/Akt和ERK信号通路广泛参与细胞的有丝分裂、代谢运动、生存、凋亡和分化等过程。其在血管新生中的作用也越来越受到重视。所以,我们设想Rap1GAP是通过PI3K/Akt和ERK信号通路来实现其在血管新生中的作用。本研究的目的就在于研究Rap1GAP在血管新生中的作用机理。研究方法首先通过人脐静脉获得内皮细胞后对其培养、扩增。利用Fugene 6转染脐静脉内皮细胞,使其高表达Rap1GAP。然后通过BrdU-ELISA方法检测转染Rap1GAP后,内皮细胞增殖变化情况,利用Transwell小室测定内皮细胞的迁移能力,通过体外管腔形成实验检测微血管样结构的形成情况。为了研究Rap1GAP对内皮细胞发挥作用的机理及信号传导通路,通过免疫印迹的方法检测内皮细胞转染Rap1GAP后ERK, Akt磷酸化的变化情况。同时进一步观察ERK和Akt信号传导通路特异拮抗剂对内皮细胞细胞增殖、迁移的影响。最后,对转染有Rap1GAP的HUVECs给予外源性VEGF刺激后观察其增殖、Cyclin D1以及ERK、Akt磷酸化的变化情况,从而研究Rap1GAP对VEGF促进内皮细胞增殖是否影响及作用机制。实验结果 1高表达的Rap1GAP能够抑制内皮细胞的增殖、迁移和体外管腔形成能力。 2 Rap1GAP能够抑制Rap1/Akt和Rap1/ERK信号通路,进而抑制内皮细胞的增殖、迁移. 3 Rap1GAP高表达有抑制VEGF诱导的内皮细胞增殖的效应,可能是通过Rap1/ERK和Rap1/Akt通路抑制细胞周期来实现对此效应的调节。实验结论 Rap1GAP能够抑制内皮细胞的增殖、迁移,通过Akt和ERK信号通路来发挥该作用。
[Abstract]:Purpose of research Rap1 is a member of small G protein Ras family. In vitro, it has been proved that Rap1 plays many roles in endothelial cells. Rap1 plays a major role in the regulation of endothelial cell adhesion formation, permeability and other functions. Recent studies have shown that Rap1 in endothelial cells is not limited to cell adhesion, but also regulates the response of endothelial cells to some factors that promote angiogenesis. In other words, Rap1 may be a positive angiogenesis regulator in vivo. Rap1GAP is the GTP activator of small G protein Rap1, which can accelerate the transition of Rap1 from the active form of GTP binding to the deactivated form of GDP binding. Some researchers have found that Rap1GAP inactivates Rap1 and inhibits Rap1-mediated angiogenesis. But there are some experimental results that do not support this conclusion. Therefore, it is necessary to further study the role of Rap1GAP in angiogenesis. Revealing the role of Rap1GAP in angiogenesis is helpful to further understand the mechanism of angiogenesis and provide a new and specific breakthrough point for angiogenesis related diseases. Angiogenesis is the process by which the microvessels sprout from the already existing vascular beds and gradually form new branches and capillary plexus, which consist of a series of coordinated and closely coordinated processes. Many factors are involved in its regulation. PI3K / Akt and ERK signaling pathway are widely involved in mitosis, metabolic movement, survival, apoptosis and differentiation. More and more attention has been paid to its role in angiogenesis. Therefore, we assume that Rap1GAP plays a role in angiogenesis through PI3K/Akt and ERK signaling pathways. The purpose of this study is to study the mechanism of Rap1GAP in angiogenesis. Research method First, endothelial cells were obtained through human umbilical vein and then cultured and amplified. Umbilical vein endothelial cells (HUVEC) were transfected with Fugene 6 to overexpression Rap1 GAP. Then the changes of endothelial cell proliferation after transfection of Rap1GAP were detected by BrdU-ELISA method, the migration ability of endothelial cells was measured by Transwell chamber, and the formation of microvessel like structure was detected by in vitro lumen formation experiment. In order to study the mechanism and signal transduction pathway of Rap1GAP on endothelial cells, the phosphorylation of ERK and Akt in endothelial cells transfected with Rap1GAP was detected by Western blotting. The effects of ERK and Akt signal transduction pathway specific antagonists on the proliferation and migration of endothelial cells were also observed. Finally, the phosphorylation of cyclin D1 and ERKN Akt in HUVECs transfected with Rap1GAP was observed after stimulation with exogenous VEGF, and the effect of Rap1GAP on the proliferation of endothelial cells induced by VEGF and its mechanism were studied. Experimental results 1 High expression of Rap1GAP could inhibit endothelial cell proliferation, migration and in vitro lumen formation. 2 Rap1GAP can inhibit Rap1/Akt and Rap1/ERK signaling pathway, and then inhibit the proliferation and migration of endothelial cells. 3High expression of Rap1GAP can inhibit the proliferation of endothelial cells induced by VEGF, which may be mediated by inhibition of cell cycle by Rap1/ERK and Rap1/Akt pathway. Experimental conclusion Rap1GAP inhibits the proliferation and migration of endothelial cells through Akt and ERK signaling pathways.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R329

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