高切高脂通过VEGF-VEGFR2信号通路调控血管新生参与动脉粥样易损斑块形成

发布时间：2018-06-06 23:05

本文选题：动脉粥样硬化易损斑块 + 血管新生　；参考：《重庆大学》2015年博士论文

【摘要】：目前的研究和临床表明,位列全球死亡人数的前两位的疾病分别为缺血性心脏病和脑血管疾病,高达死亡人数的21.9%。伴随全国人民生活水平的提高,心脑血管疾病已经跃居为我国的第一位杀手。冠状动脉粥样硬化发病最显著的特征是冠状动脉的不稳定斑块破裂,形成血栓堵塞冠状动脉。因此,对预防冠状动脉粥样硬化最重要的手段是早期预判和治疗不稳定性易损斑块。冠状动脉粥样硬化病理学研究表明,易损斑块具有以下组织病理学特征:在血管狭窄处容易形成较大的斑块;斑块中有薄的纤维帽;炎症细胞和大的脂核浸润;斑块内有较多的新生血管。动脉粥样硬化易损斑块内出现的病理性新生血管,能加速粥样硬化病变的发展,从而诱发斑块破裂脱落,形成血栓堵塞血管,促进其急性心血管事件的发生。研究报道指出,AS(Atherosclerosis)易损斑块的形成和破裂的病理原因,一直没能完全阐述明白,尤其是在高脂和变化的应力场环境下的趋势更是无法解释清楚。而有观点指出,滋养血管增生以及在斑块内高密度富集,是其核心关键点。此时出现的滋养血管,其功能和结构不完整,尤其外膜的不稳定,极易发生侧漏,从而诱发斑块内出血和破损出现。因而本文从斑块内血管新生入手,探索AS易损斑块形成、发展的病因;引入高脂环境和高切应力因素,研究损伤血管在复杂环境下,斑块内血管新生与其病变过程之间的关系。VEGF(Vascular Endothelial Growth Factor)是特异性和作用最强的血管新生因子,参与了多种体内的病理性血管新生,在肿瘤和斑块血管新生中起着关键调控蛋白的作用。在肿瘤血管新生的研究中,已经系统的探索了VEGF及下游通路蛋白在肿瘤病变中的作用机理。然而在AS易损斑块的研究中,只是证实了VEGF参与了斑块的病变和血管新生过程,但是对于VEGF影响斑块血管新生的机理、下游调控蛋白在AS斑块中的作用、以及与AS易损斑块的高危因素高脂高切应力之间的关系研究,还无准确的定论。基于此,本文通过对血管新生经典通路VEGF-VEGFR2-FAK(Focal adhesion kinase)/paxillin在oxLDL(Oxidized low density liporprotein)和高切应力介导的血管新生中的作用研究,明确了VEGF及下游通路蛋白在AS易损斑块形成中的作用机制,为临床治疗AS贡献了新的基因靶点。本论文中,我们使用的动物模型为小鼠左颈总动脉套环模型。在此基础上给小鼠饲喂高脂饲料(1%胆固醇和5%猪油),进而研究斑块的组成、血管新生数量。通过血管新生抑制剂(TNP-470)对模型组注射治疗,研究治疗组斑块的形成和血管新生情况。从动物模型上,证明血管新生能促进动脉粥样硬化易损斑块的形成。在正常组和模型组血管中分别对血管新生相关基因vegf、cd31(plateletendothelialcelladhesionmolecule-1)进行免疫染色,发现模型组中vegf和cd31表达都集中在斑块的肩部和纤维帽区域(较正常组)。并通过合成vegfsirna、vegfr1sirna、vegfr2sirna、vegf重组蛋白对高脂高切应力影响下的内皮细胞刺激,观察下游通路蛋白fak/paxillin的活化(磷酸化),以及对内皮细胞迁移和管腔形成的影响,来探索高脂高切应力介导下的斑块血管新生的信号传导途径。主要研究内容和结果如下:①构建小鼠左颈动脉套环模型,右侧颈动脉进行假手术对照。单一套环模型只能刺激内膜增生,不诱导易损斑块的形成。其中增生的体积在高低切应力区域基本一致。加入高脂因素,左颈动脉高切应力区域(狭窄近心端)出现大量不稳定斑块,低切应力区域(远心端)无斑块形成,仅有一定内膜增生。而正常组血管,在高脂环境下,其血管壁有少量脂肪颗粒沉积和内膜增生。通过he染色,显示高切应力区域易损斑块内的新生血管主要为外膜不完整、内皮细胞有凋亡趋势、管腔内有大量淋巴细胞浸润的易泄漏微血管。而tnp-470治疗组,血管内膜增生减弱,易损斑块中的血管新生有所减弱,斑块面积减少。②通过对小鼠颈动脉进行血管新生相关蛋白vegf、cd31免疫染色。发现在正常组中,血管无vegf、cd31的阳性表达。而模型组高切应力组中,vegf、cd31在斑块的肩部和纤维帽区域都存在阳性高表达。研究显示,vegf参与了斑块血管新生的病理过程,从而为我们选择vegf作为关于血管新生的研究靶点提供了依据。③通过用oxldl和切应力分别刺激huvecs(humanumbilicalvascularendothelialcell),研究两者对huvecs迁移和管腔形成的影响。发现正常组内皮细胞无迁移和管腔形成。而oxldl组内皮细胞迁移和管腔形成明显,其中最佳浓度为20μg/ml。高切应力组内皮细胞迁移和管腔形成明显,最佳压力为25dyn/cm2。研究显示,oxldl和高切应力能促进内皮细胞功能主导的血管新生。④通过合成vegfsirna、vegf重组蛋白对高脂高切应力影响下的内皮细胞刺激,研究vegf在oxldl和高切应力介导的血管新生中的作用。实验结果表明,vegf的过表达,能促进huvecs迁移和管腔形成;同时高脂高切应力都能促进huvecs中vegf及下游受体(vegfr1、vegfr2)的过表达;vegfsirna组内皮细胞用oxldl和高切应力刺激,内皮细胞的迁移和管腔形成明显减少(较oxldl和高切应力组)。研究显示,vegf是oxldl和高切应力调控血管新生的关键蛋白。⑤通过合成vegfr1sirna、vegfr2sirna、vegf重组蛋白、vegfsirna对高脂高切应力影响下的内皮细胞刺激,研究vegf-vegfr2-fak/paxillin信号通路在ox LDL和高切应力介导的血管新生中的作用。实验结果说明,VEGF能调控VEGFR2下游蛋白FAK/paxillin的磷酸化;oxLDL和高切应力通过VEGF调控VEGFR2-FAK/paxillin下游信号通路的表达;同时VEGFR2能调控下游蛋白FAK/paxillin的磷酸化;VEGFR2/VEGFR1是VEGF调控血管新生的关键因子。因此,在高脂高切应力调控的血管新生中,VEGF-VEGFR2-FAK/paxillin信号通路起着关键调控通路作用。综上所述,通过本研究工作,我们阐明了血管新生在动脉粥样易损斑块中的作用,同时验证了VEGF-VEGFR2-FAK/paxillin信号通路在高脂高切应力所调控的动脉粥样硬化斑块内血管新生中的作用。
[Abstract]:Current research and clinical studies show that the top two diseases in the world are ischemic heart disease and cerebrovascular disease, and the 21.9%. of the death toll is accompanied by the improvement of the National People's living standard. Cardiovascular and cerebrovascular diseases have leaped into the first killer in our country. The most significant characteristic of coronary atherosclerosis is the characteristic of coronary atherosclerosis. The most important means of preventing coronary atherosclerosis is early prediction and treatment of unstable vulnerable plaques. The pathological study of coronary atherosclerotic plaque shows that vulnerable plaques have the following histologic features: easy to form in vascular stenosis. Large plaques; thin fibrous caps in plaques; inflammatory cells and large fat nuclei; there are more neovascularization in the plaque. Atherosclerotic Vulnerable Atherosclerotic neovascularization can accelerate the development of atherosclerotic lesions, resulting in plaque rupture and abscission, thrombosis and blockage of blood vessels, and the promotion of acute cardiovascular events. The report points out that the pathological causes of the formation and rupture of vulnerable plaques of AS (Atherosclerosis) have not been fully explained, especially in the high fat and changing stress field environment, and it is pointed out that the key point is to increase the growth of the nourishing blood vessels and to enrich the density in the plaque. In this case, the function and structure of the nourishing blood vessels are incomplete, especially the instability of the outer membrane and the occurrence of hemorrhage and breakage in the plaque. Therefore, this paper, starting with the angiogenesis in the plaque, explores the formation of AS vulnerable plaques, the cause of development, the introduction of high fat environment and high shear stress factors, and the complexity of the damage vessels in complex research. .VEGF (Vascular Endothelial Growth Factor) is a specific and strongest angiogenic factor in the environment, which is a specific and most powerful angiogenic factor. It participates in a variety of pathological angiogenesis in the body and plays the role of key regulatory proteins in tumor and plaque angiogenesis. The mechanism of VEGF and downstream pathway proteins in tumor lesions has been systematically explored. However, in the study of AS vulnerable plaques, it is only confirmed that VEGF is involved in plaque lesion and angiogenesis, but the mechanism that VEGF affects plaque angiogenesis, the role of the downstream modulation protein in the AS plaque, and the vulnerability to AS There is no accurate conclusion of the relationship between high risk of plaque and high fat and high shear stress. Based on this, this paper studies the role of VEGF-VEGFR2-FAK (Focal adhesion kinase) /paxillin in the angiogenesis in oxLDL (Oxidized low density liporprotein) and high shear stress, and makes clear the VEGF and downstream. The mechanism of pathway protein in the formation of AS vulnerable plaque contributes to the new gene target for clinical treatment of AS. In this paper, we use the animal model of left neck total artery ring model in mice. On this basis, we feed mice with high fat diet (1% cholesterol and 5% lard), and then study the composition of the plaque, the number of angiogenesis, and the blood flow through blood. The formation of plaque and angiogenesis in the treatment group were studied by injection of neonatal inhibitor (TNP-470) to the model group. From the animal model, it was proved that angiogenesis could promote the formation of vulnerable plaque in atherosclerosis. In the normal group and the model group, the neovascularization gene VEGF, CD31 (plateletendothelialcelladhesionmo), respectively, was used in the normal and model group. Lecule-1) immunohistochemical staining showed that the expression of VEGF and CD31 in the model group was concentrated in the shoulder and the fiber cap region of the plaque (the normal group). The activation of the downstream pathway protein fak/paxillin (phosphorylation) was observed through the synthesis of vegfsirna, vegfr1sirna, vegfr2sirna, and VEGF recombinant protein on the endothelial cells under the influence of high lipid high shear stress. The main research contents and results are as follows: (1) the model of the left carotid artery in mice is constructed and the right carotid artery is controlled by the false operation. The single ring model can only stimulate the hyperplasia of the intima, and it does not induce easily. The volume of the lesion was basically the same in the area of high and low shear stress. A large number of unstable plaques appeared in the high shear stress region (narrow near the heart end) in the left carotid artery, and the low shear stress region (Yuan Xinduan) had no plaque formation and only a certain intimal hyperplasia. He staining showed that the neovascularization in the vulnerable plaque of the high shear stress area was mainly the incomplete outer membrane, the endothelial cells had the tendency of apoptosis, and the intravascular infiltration of lymphocytes were easy to leak microvessels in the lumen. In the TNP-470 treatment group, the intravascular membrane hyperplasia was weakened, and the angiogenesis in vulnerable plaque was found. The plaque area decreased. (2) the vascular neovascularization related protein VEGF, CD31 immunostaining was performed on the carotid artery in mice. It was found that in the normal group, the blood vessels were not VEGF and CD31 positive. While in the high shear stress group, VEGF and CD31 were positive and high in the shoulder and fiber cap regions of the plaque. The study showed that VEGF was involved in the plaque blood. The pathological process of neovascularization provides a basis for our selection of VEGF as a target for angiogenesis. (3) the effect of both oxLDL and shear stress on HUVECs (humanumbilicalvascularendothelialcell), respectively, on the migration of HUVECs and the formation of the lumen in the lumen. The migration of endothelial cells and the formation of the lumen in the oxLDL group were obvious. The best concentration was the migration of endothelial cells and the formation of the lumen in the high shear stress group of 20 mu g/ml.. The best pressure was the 25dyn/cm2. study. The oxLDL and high shear stress could promote the angiogenesis of endothelial cells. (4) through the synthesis of vegfsirna, the recombinant protein of VEGF should be highly cut to high fat. The effect of VEGF on the angiogenesis in oxLDL and high shear stress was studied under the influence of force. The results showed that the overexpression of VEGF could promote the migration of HUVECs and the formation of the lumen, and the high fat and high shear stress could promote the overexpression of VEGF and the downstream receptor (VEGFR1, VEGFR2) in HUVECs; the vegfsirna group of endothelial cells used oxLDL. The migration of endothelial cells and the formation of endothelium were significantly reduced (compared with oxLDL and high shear stress groups). The study showed that VEGF was a key protein in the regulation of angiogenesis by oxLDL and high shear stress. 5. Through the synthesis of vegfr1sirna, vegfr2sirna, VEGF recombinant protein, and vegfsirna to the endothelial cell stimulation under the influence of high fat and high shear stress, study veg The role of f-vegfr2-fak/paxillin signaling pathway in ox LDL and high shear stress mediated angiogenesis. The experimental results show that VEGF regulates the phosphorylation of the downstream protein FAK/paxillin of VEGFR2; oxLDL and high shear stress regulate the expression of the downstream signal pathway of VEGFR2-FAK/paxillin through VEGF; and VEGFR2 can regulate the FAK/paxillin downstream protein. Phosphorylation; VEGFR2/VEGFR1 is a key factor in the regulation of angiogenesis in VEGF. Therefore, the VEGF-VEGFR2-FAK/paxillin signaling pathway plays a key regulatory pathway in the angiogenesis of high fat and high shear stress. In this study, we elucidated the role of new vascular neovascularization in atherosclerotic plaque. The role of VEGF-VEGFR2-FAK/paxillin signaling pathway in angiogenesis in atherosclerotic plaques regulated by high fat and high shear stress.
【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R543.5

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