本文選題：Rap1GAP + Rap1　； 參考：《華中科技大學(xué)》2011年博士論文

【摘要】：研究目的 Rap1是小分子G蛋白Ras家族成員之一,體外的實驗已經(jīng)證明Rap1在細(xì)胞內(nèi)發(fā)揮多種作用。Rap1對內(nèi)皮細(xì)胞的作用主要是參與對內(nèi)皮細(xì)胞間粘連形成、通透性等功能的調(diào)節(jié)。最近的研究表明,在內(nèi)皮細(xì)胞中,Rap1不僅僅局限于細(xì)胞粘連,而且可以調(diào)節(jié)內(nèi)皮細(xì)胞對一些促進(jìn)血管新生刺激因素的反應(yīng)。換句話說,Rap1在體內(nèi)可以作為一種正性血管新生調(diào)節(jié)因素。 Rap1GAP是小分子G蛋白Rap1的GTP酶激活蛋白,它可以加速Rap1由與GTP結(jié)合的活化形式轉(zhuǎn)變?yōu)榕cGDP結(jié)合的去活化形式的過程。一些研究者發(fā)現(xiàn)Rap1GAP可以失活Rap1,抑制Rap1介導(dǎo)的血管新生。但也有一些實驗結(jié)果并不支持這一結(jié)論。因此,很有必要進(jìn)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進(jìn)一步了解血管新生的機理,并且能為與血管新生相關(guān)疾病提供一個新的、特異的研究切入點。 血管新生是微血管從已經(jīng)存在的血管床以出芽的方式長出,并逐漸形成新的血管分支及毛細(xì)血管叢的過程,它由一系列相互協(xié)調(diào)密切配合的過程來組成的,許多因素參與了對其的調(diào)節(jié)。PI3K/Akt和ERK信號通路廣泛參與細(xì)胞的有絲分裂、代謝運動、生存、凋亡和分化等過程。其在血管新生中的作用也越來越受到重視。所以,我們設(shè)想Rap1GAP是通過PI3K/Akt和ERK信號通路來實現(xiàn)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機理。 研究方法 首先通過人臍靜脈獲得內(nèi)皮細(xì)胞后對其培養(yǎng)、擴增。利用Fugene 6轉(zhuǎn)染臍靜脈內(nèi)皮細(xì)胞,使其高表達(dá)Rap1GAP。然后通過BrdU-ELISA方法檢測轉(zhuǎn)染Rap1GAP后,內(nèi)皮細(xì)胞增殖變化情況,利用Transwell小室測定內(nèi)皮細(xì)胞的遷移能力,通過體外管腔形成實驗檢測微血管樣結(jié)構(gòu)的形成情況。 為了研究Rap1GAP對內(nèi)皮細(xì)胞發(fā)揮作用的機理及信號傳導(dǎo)通路,通過免疫印跡的方法檢測內(nèi)皮細(xì)胞轉(zhuǎn)染Rap1GAP后ERK, Akt磷酸化的變化情況。同時進(jìn)一步觀察ERK和Akt信號傳導(dǎo)通路特異拮抗劑對內(nèi)皮細(xì)胞細(xì)胞增殖、遷移的影響。 最后,對轉(zhuǎn)染有Rap1GAP的HUVECs給予外源性VEGF刺激后觀察其增殖、Cyclin D1以及ERK、Akt磷酸化的變化情況,從而研究Rap1GAP對VEGF促進(jìn)內(nèi)皮細(xì)胞增殖是否影響及作用機制。 實驗結(jié)果 1高表達(dá)的Rap1GAP能夠抑制內(nèi)皮細(xì)胞的增殖、遷移和體外管腔形成能力。 2 Rap1GAP能夠抑制Rap1/Akt和Rap1/ERK信號通路,進(jìn)而抑制內(nèi)皮細(xì)胞的增殖、遷移. 3 Rap1GAP高表達(dá)有抑制VEGF誘導(dǎo)的內(nèi)皮細(xì)胞增殖的效應(yīng),可能是通過Rap1/ERK和Rap1/Akt通路抑制細(xì)胞周期來實現(xiàn)對此效應(yīng)的調(diào)節(jié)。實驗結(jié)論 Rap1GAP能夠抑制內(nèi)皮細(xì)胞的增殖、遷移,通過Akt和ERK信號通路來發(fā)揮該作用。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王建民，劉蔭秋，，賴西南;正常臍靜脈離體后不同時間內(nèi)皮某些物質(zhì)含量的變化[J];第三軍醫(yī)大學(xué)學(xué)報;1995年01期

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