Hes-1介导VEGF促进OPN表达在动脉粥样硬化发生发展过程中的作用及分子机制研究

发布时间：2018-07-23 18:09

【摘要】：研究背景和目的:动脉粥样硬化是导致心血管疾病的最关键致病因素。新生血管形成是粥样硬化斑块特征之一,它可以引发易损斑块的发展,增加斑块破裂的风险。但是,血管新生发生及发展的调控机制直至今日未明确。DNA结合蛋白(hairy enhancer of split,Hes-1)是一类DNA结合蛋白,因其结构中含有α螺旋-环-螺旋(α HLH)而极具特征性。这一物质可结合在特定分化效应基因的启动子上,从而发挥介导细胞分化的抑制信号,并参与血管细胞发育、分化以及及新生血管形成等重要生理过程。Hes-1的调节转录在心血管以及平滑肌细胞的分化、血管的形成、动静脉血管细胞的凋亡以及血管成形的过程中起到至关重要的作用。目前的一些研究已表明,Hes-1涉及了调节内皮细胞(EC)的增殖、分化和凋亡的过程。但是,直至今日,关于HES-1是否参与到不稳定斑块中血管生成的过程中仍不清楚。骨桥蛋白(osteopontin,OPN)是一种分泌型糖基化磷蛋白,存在于细胞外基质中,其作用是参与介导细胞的黏附、增殖以及迁移,对于调节血管重构起重要作用。初步研究表明,OPN不仅促进动脉粥样硬化,还促进了新生血管的生成。在内皮发生损伤后,平滑肌细胞出现一系列去分化、迁移、增殖的过程,在这些过程中OPN通过介导细胞与细胞、细胞与基质的相互作用进而发挥了促进血管重塑的作用。虽然OPN调控机制在血管生成过程中仍不清楚,有研究显示Hes-1过表达抑制成骨细胞OPN转录,这表明Hes-1可能是OPN潜在调节因子。在动脉粥样硬化斑块中,内皮细胞以及炎性细胞可以分泌产生血管内皮生长因子(VEGF),其可作为始动因子参与是斑块以及血管生成,与此同时Hes-1也参与了 VEGF的信号转导。除了 hes-1,越来越多的研究发现VEGF诱导OPN的表达。然而,Hes-1是否通过调节OPN进而影响VEGF诱导的动脉粥样硬化斑块中新生血管生成尚未阐明。本课题研究拟通过分别检测Hes-1和OPN在动脉粥样硬化组织中的表达情况,进而分析这两种物质的表达与相关病理参数之间的内在联系,与此同时通过进行体外模型的实验,研究并揭示Hes-1和OPN在AS疾病的发生、发展过程中的所发挥的作用和分子学相关机制。方法1.通过使用组织基因芯片来分析Hes-1和OPN为粥样硬化动脉组织对比正常动脉组织标本中的差异性表达基因;2.荧光定量qPCR、免疫组织化学(IHC)和免疫荧光(immunofluorescence)检测粥样硬化动脉组织与正常动脉组织样本中Hes-1和OPN的表达,并分析其的表达量、定位;3.通过体外VEGF刺激的方法,利用免疫印迹试验(Westernblot)检测Hes-1和OPN表达情况;4.构筑建造Hes-1过表达型的载体,并使用合成siRNA对Hes-1的表达进行干扰,对脐静脉内皮细胞进行瞬时转染,利用western blot方法检测Hes-1对OPN的调控作用;5.构建OPN过表达载体,合成siRNA干扰OPN的表达,通过基质胶成管实验检测VEGF-Hes-1-OPN对血管生成的影响。结果基因芯片、RT-PCR以及IHC检测的结果显示:在动脉粥样硬化斑块的样本中,Hes-1的表达水平显著性降低,但是OPN的表达却明显增强。同时,免疫荧光结果显示,Hes-1和OPN均在斑块中新生血管的内皮中表达。体外细胞实验表明,在VEGF的刺激下,脐静脉内皮细胞中Hes-1呈时间及剂量依赖性降低,OPN呈时间及剂量依赖性升高。将Hes-1基因表达进行敲低处理后,可以增强OPN的表达以及VEGF诱导性下的血管生成。与此相反的是,如Hes-1基因过表达则会对OPN的表达起抑制作用并对VEGF诱导的血管生成起负性调节作用。结论动脉粥样斑块组织中Hes-1抑制了 OPN的表达,并在此基础上抑制VEGF诱导的血管生成的发生及发展。因此,恢复Hes-1的表达、抑制OPN基因可作为预防不稳定斑块形成的重要举措。
[Abstract]:Background and objective: atherosclerosis is the most important cause of cardiovascular disease. Neovascularization is one of the characteristics of atherosclerotic plaque. It can cause the development of vulnerable plaque and increase the risk of plaque rupture. However, the regulation mechanism of angiogenesis and development of angiogenesis is not clear to.DNA binding protein until today (hairy Enhancer of split, Hes-1) is a class of DNA binding proteins which are characterized by its structure containing alpha spiral loop helix (alpha HLH). This substance can be combined with the promoter of a specific differentiation effect gene to play an inhibitory signal to mediate cell differentiation, and to participate in the development of vascular cells, differentiation, and the formation of neovascularization. Regulatory transcription of.Hes-1 is essential for the differentiation of cardiovascular and smooth muscle cells, the formation of blood vessels, the apoptosis of arteriovenous cells and the process of angioplasty. Some studies have shown that Hes-1 involves the process of regulating the proliferation, differentiation and apoptosis of endothelial cells (EC), but to this day, It is not clear whether HES-1 is involved in angiogenesis in unstable plaques. Osteopontin (OPN) is a kind of secreted glycosylated phosphoprotein, which exists in the extracellular matrix. Its role is to mediate cell adhesion, proliferation and migration, and plays an important role in regulating vascular remodeling. Preliminary studies have shown that OPN does not It only promotes atherosclerosis and promotes the formation of new blood vessels. After endothelial damage, the smooth muscle cells have a series of processes of dedifferentiation, migration and proliferation. In these processes, OPN plays a role in promoting vascular remodeling by mediating the interaction between cells and cells, cells and the matrix. Although the mechanism of OPN regulation is in blood Guan Shengcheng's process is still unclear. Studies have shown that overexpression of Hes-1 inhibits osteoblast OPN transcription, which suggests that Hes-1 may be a potential regulator of OPN. In atherosclerotic plaques, endothelial cells and inflammatory cells can secrete the production of vascular endothelial growth factor (VEGF), which can be involved in plaque and angiogenesis as a initiating factor. At the same time, Hes-1 also participates in the signal transduction of VEGF. In addition to HES-1, more and more studies have found that VEGF induces the expression of OPN. However, Hes-1 has not been elucidated by regulating OPN and thus affecting the angiogenesis in VEGF induced atherosclerotic plaques. This study is to detect Hes-1 and OPN in atherosclerotic atherosclerosis by detecting Hes-1 and OPN respectively. And then analyze the expression in the tissue, and then analyze the internal relationship between the expression of the two substances and the related pathological parameters. At the same time, through the experiment of the model in vitro, the role of Hes-1 and OPN in the development of AS disease, the role of the molecular mechanism in the development process and the molecular mechanism are revealed. Method 1. through the use of the tissue gene chip To analyze the differential expression genes of Hes-1 and OPN in atherosclerotic arterial tissue compared with normal arterial tissue, 2. fluorescence quantitative qPCR, immunohistochemistry (IHC) and immunofluorescence (immunofluorescence) were used to detect the expression of Hes-1 and OPN in atherosclerotic arterial tissue and normal arterial tissue samples, and to analyze the expression and location of 3.. The expression of Hes-1 and OPN was detected by the method of VEGF stimulation in vitro, and the expression of Hes-1 and OPN was detected by Western blot test (Westernblot); 4. the Hes-1 overexpression vector was constructed and siRNA was used to interfere with the expression of Hes-1, and the umbilical vein endothelial cells were transiently transfected, and Western blot method was used to detect Hes-1 to OPN, and 5. to construct O. PN overexpressed the expression vector, synthesized the expression of siRNA interfering OPN, and detected the effect of VEGF-Hes-1-OPN on angiogenesis. Results the results of gene chip, RT-PCR and IHC showed that the expression level of Hes-1 was significantly decreased in the samples of atherosclerotic plaque, but the expression of OPN was obviously enhanced. Meanwhile, the immune system was immune. The fluorescence results showed that both Hes-1 and OPN were expressed in the endothelial cells of the neovascularization in the plaque. In vitro cell experiments showed that under the stimulation of VEGF, the Hes-1 in the umbilical vein endothelial cells decreased in time and dose dependence, and the OPN was increased in time and dose dependence. The expression of Hes-1 gene and the VEGF could enhance the expression of OPN and VEGF. In contrast, the overexpression of Hes-1 gene can inhibit the expression of OPN and negatively regulate the angiogenesis induced by VEGF. Conclusion Hes-1 inhibits the expression of OPN in the atherosclerotic plaque tissue and inhibits the occurrence and development of angiogenesis induced by VEGF. The expression of compound Hes-1 and inhibition of OPN gene can be used as an important measure to prevent the formation of unstable plaque.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R543.5

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