轴突导向分子Sema7A参与小鼠动脉粥样硬化发生发展机制的研究
发布时间：2018-09-01 12:08
【摘要】：动脉粥样硬化是引发心血管疾病的主要原因。近年来,动脉粥样硬化被认为是一种系统性免疫炎症的疾病,先天性免疫与获得性免疫均参与其。损伤早期形态学变化即脂质条纹,是一种严重的代谢和免疫损伤,表现为血管张力异常,炎症,细胞和内皮功能障碍。随后损伤形成斑块继续生长,最终凸入管腔或发生破裂。当脂质斑块破裂后,形成的动脉血栓会导致急性冠脉综合征(ACS)、心肌梗死或脑中风等恶性临床事件,对人类的健康危害极大。但其发病机制目前仍不是十分清楚,寻找有效药物对该疾病的治疗迫在眉睫。Semaphrin家族是最初以参与轴突导向发现的一类蛋白分子家族,该类蛋白与组织器官的形成,血管生成与免疫细胞调控密切相关。Semaphrin家族包括20多个成员共分成8个亚类,Semaphrin7A(Sema7A)是跨膜的糖基化磷脂酰基醇蛋白,属于第七类轴突导向分子家族,Sema7A能够与β1整合素结合从而介导嗅觉神经的生长[1]并启动激活由T细胞介导的免疫应答。Sema7A蛋白可以通过与α1β1结合刺激巨噬细胞和单核细胞产生诸如IL-8,IL-6,IL-1β以及TNF-α等炎症因子。近期另有多篇研究发现,Sema7A在肝纤维化,神经损伤,肺纤维化,皮肤炎症,角膜炎症和多种硬化症中均有发挥促炎作用。尽管目前我们得知Sema7A参与多种生理性过程及病理性疾病中,但Sema7A是否参与动脉粥样硬化疾病的发生尚无报道。Sema7A及其受体在T淋巴细胞、单核细胞,树突状细胞以及血小板中均有表达,而这些细胞恰恰是与动脉粥样硬化的发生有着密切关系的细胞种类。且有文献证明内皮细胞上的Sema7A可以诱导缺氧过程中中性粒细胞向内皮下层的迁移浸润过程,这也提示Sema7A或在动脉粥样硬化过程中也可以介导炎症细胞向内膜下层的迁移浸润。因此我们着手研究Sema7A在动脉粥样硬化过程中的作用,并发现Sema7A敲除的情况下可以减少载脂蛋白E敲除小鼠的斑块形成并减缓动脉粥样硬化的发展。目的:本文旨在探讨轴突导向分子Sema7A是否能够影响动脉粥样硬化脂质斑块形成,最终影响动脉粥样硬化的发展。研究方法:1.利用Apo E-/-小鼠喂食高脂饲料1周,4周,12周,通过RNA以及蛋白水平检测Sema7A及受体在动脉粥样硬化发展过程中的表达情况。2.制备Sema7A-/-Apo E-/-双敲除小鼠。3.通过对载脂蛋白E(Apo E)敲除小鼠(以下简写为Apo E-/-小鼠)喂食高脂饲料(high-fat diet,以下简写为HFD),建立动脉粥样硬化小鼠模型。4.Sema7A-/-Apo E-/-双敲除小鼠与Sema7A+/+Apo E-/-单敲除小鼠,,在喂食高脂饲料12周前后的体重对比,检测Sema7A存在与否对动脉粥样硬化小鼠的体重产生影响。并通过眼眶后静脉丛抽取高脂血症小鼠的静脉血,分离血清,检测比较Sema7A-/-Apo E-/-双敲除小鼠与Sema7A+/+Apo E-/-单敲除小鼠血浆中的血脂含量,分析Sema7A对高脂血症状态下小鼠血脂含量的影响。5.当喂食HFD 12周后,Apo E-/-小鼠能够形成稳定的动脉粥样硬化斑块,钝性分离得到小鼠整只主动脉,通过苏丹红染色,对比Sema7A-/-Apo E-/-双敲除小鼠与Sema7A+/+Apo E-/-单敲除小鼠斑块的分布与大小,研究Sema7A是否影响Apo E-/-小鼠冠状及胸主动脉中脂质斑块的形成。并通过对比两组小鼠斑块内细胞种类以及分布的不同,研究Sema7A存在与对斑块内细胞成分的影响。6.取形成稳定斑块的小鼠的主动脉组织,通过提取组织RNA,逆转录并通过定量PCR方法检测巨噬细胞活化亚型的相应细胞因子表达,进而分析巨噬细胞的极化情况。7.动脉粥样硬化模型构建成功后,分离全血血清,比较Sema7A-/-Apo E-/-双敲除小鼠与Sema7A+/+Apo E-/-单敲除小鼠血浆中T细胞活化分泌的细胞因子的水平以及炎症因子的水平,检测Sema7A对高脂血症状态下小鼠炎症因子含量的影响。结果:1.在喂食HFD期间,Apo E-/-小鼠主动脉内Sema7A的表达呈规律变化。与喂食普通饲料相比,喂食HFD可以明显上调主动脉组织中Sema7A的表达,并且在喂食一周时Sema7A的表达量达到最高。2.成功制备了Sema7A-/-Apo E-/-双敲除小鼠。3.小鼠在喂食12周HFD后其冠状及胸主动脉中产生了大量脂质斑块,Sema7A-/-Apo E-/-双敲除小鼠斑块大小较Sema7A+/+Apo E-/-单敲除小鼠降低了51.18%(Sema7A-/-Apo E-/-:5.01±1.04%,Sema7A+/+Apo E-/-:10.26±1.15%,P=0.0024)。4.动脉粥样硬化小鼠模型建立后,与Sema7A+/+Apo E-/-单敲除小鼠相比,Sema7A-/-Apo E-/-双敲除小鼠的斑块内巨噬细胞以及免疫细胞T细胞,树突状细胞的含量有比较明显的降低。5.通过主动脉根部斑块切片染色发现,与Apo E-/-Sema7A+/+单敲除小鼠相比,Apo E-/-Sema7A-/-双敲除小鼠的斑块内胶原含量明显上调(Sema7A-/-Apo E-/-:88.37±0.75%;Sema7A+/+Apo E-/-:82.00±1.38%,P0.0022),-/-+/+,且斑块的易破损系数明显降低,斑块的稳定性明显增强。6.动脉粥样硬化小鼠模型建立后,与-Sema7A+/+Apo E-/-单敲除小鼠相比,Sema7A-/-Apo E-/-双敲除小鼠主动脉组织中1型巨噬细胞M1有明显的降低,2型巨噬细胞的含量有一定的升高趋势。7.通过动脉粥样硬化小鼠的血浆中血脂含量以及免疫细胞相关炎症因子的检测,我们发现,相较于单敲小鼠,双敲除小鼠的炎症因子的水平明显降低且血浆中促炎T细胞表达明显下调。结论:我们阐明了Sema7A通过影响高脂血症状态下小鼠动脉粥样硬化斑块内巨噬细胞以及免疫细胞的数量以及免疫应答进而影响到主动脉脂质斑块的形成,从而抑制了动脉粥样硬化的发展这一机制。该研究为利用Sema7A进一步开发研究预防和治疗动脉粥样硬化疾病的潜力提供了理论基础。
[Abstract]:Atherosclerosis is a major cause of cardiovascular disease. In recent years, atherosclerosis is considered to be a systemic immune inflammatory disease, congenital immunity and acquired immunity are involved in it. Cellular and endothelial dysfunction. Subsequent plaque formation after injury continues to grow, eventually protruding into the lumen or rupture. When lipid plaque rupture, the formation of arterial thrombosis can lead to acute coronary syndrome (ACS), myocardial infarction or stroke and other malignant clinical events, great harm to human health. But the pathogenesis is still not very serious. The Semaphrin family is the first group of protein molecules to be found to be involved in axonal targeting. These proteins are closely related to organogenesis, angiogenesis and immune cell regulation. The Semaphrin family consists of more than 20 members and is divided into eight subgroups. Transmembrane glycosylated phosphatidyl alcohols belong to the seventh class of axon-directing molecules. Sema7A binds to beta-1 integrin to mediate olfactory nerve growth [1] and activate T-cell-mediated immune responses. Sema7A can stimulate macrophages and monocytes to produce such as IL-8, IL-6, IL-1 beta by binding to alpha-1 beta. Several recent studies have found that Sema7A plays an inflammatory role in liver fibrosis, nerve injury, pulmonary fibrosis, skin inflammation, corneal inflammation and a variety of sclerosis. Sema7A and its receptors are expressed in T lymphocytes, monocytes, dendritic cells and platelets, which are closely related to the development of atherosclerosis. This suggests that Sema7A may also mediate the migration and infiltration of inflammatory cells into the intimal submucosa during atherosclerosis. AIM: To investigate whether axon-directing molecule Sema7A can affect atherosclerotic lipid plaque formation and ultimately affect the development of atherosclerosis. Expression in the development of atherosclerosis. 2. Preparation of Sema7A-/-Apo E-/-double knockout mice. 3. Establishment of atherosclerotic mice model by feeding apo E-/-mice with high-fat diet (HFD). O-E-/-single knockout mice were fed with high-fat diet for 12 weeks before and after the body weight comparison, the presence of Sema7A or not on the weight of atherosclerotic mice were detected. The venous blood of hyperlipidemic mice was extracted from the retro-orbital venous plexus, and the serum was separated. Sema7A-/-Apo E-/-double knockout mice and Sema7A+/+Apo E-/-single knockout mice were compared. The effect of Sema7A on blood lipid in hyperlipidemic mice was analyzed. 5. Apo E-/- mice were able to form stable atherosclerotic plaques 12 weeks after feeding HFD. The whole aorta of mice was obtained by blunt isolation. Sema7A-/-Apo E-/-double knockout mice and Sema7A+/+Apo E-/-single knockout mice were compared by Sudan red staining. The distribution and size of plaque in knockout mice were studied to determine whether Sema7A affected the formation of lipid plaque in coronal and thoracic aortas of Apo E-/- mice. By extracting tissue RNA, reverse transcription and quantitative PCR, the expression of cytokines in the activated subtypes of macrophages was detected, and then the polarization of macrophages was analyzed. 7. After the establishment of atherosclerosis model, the whole blood serum was isolated and compared with that of Sema7A-/-Apo E-/-double knockout mice and Sema7A+/+Apo E-/-single knockout mice. Results: 1. The expression of Sema7A in the aorta of Apo E-/-mice changed regularly during feeding HFD. Compared with normal diet, HFD significantly increased S in aorta tissue. Sema7A-/-Apo E-/-double knockout mice were successfully prepared. 3. After 12 weeks of HFD, a large number of lipid plaques were produced in the coronal and thoracic aorta of the mice. The plaque size of the Sema7A-/-Apo E-/-double knockout mice was reduced by 51.18% (S 7 A+/+Apo E-/-single knockout mice) compared with the Sema7A-/+Apo E-/-single knockout mice. EM7A-/-Apo E-/-:5.01+1.04%, Sema7A+/+Apo E-/-:10.26+1.15%, P=0.0024).4. After the establishment of atherosclerotic mice model, compared with Sema7A+/+Apo E-/-single knockout mice, Sema7A-/-Apo E-/-double knockout mice plaque macrophages and immune T cells, dendritic cells content decreased significantly.5. Compared with Apo E-/-Sema7A+/+ single knockout mice, apo E-/-Sema7A-/-double knockout mice showed significantly increased collagen content in plaques (Sema7A-/-Apo E-/-:88.37+0.75%; Sema7A+/+Apo E-/-:82.00+1.38%, P 0.0022), -/-+/+, and the vulnerability coefficient of plaques was significantly reduced and the stability of plaques was significantly enhanced. After the establishment of atherosclerotic mice model, compared with - Sema7A +/+Apo E-/- single knockout mice, Sema7A-/-Apo E-/- double knockout mice aortic tissue type 1 macrophage M1 significantly decreased, type 2 macrophage content has a certain tendency to increase. 7. Through atherosclerotic mice plasma lipid content and immune cells related. We found that the levels of inflammatory factors were significantly lower and the expression of pro-inflammatory T cells in plasma was significantly lower in double knock mice than in single knock mice. Conclusion: Sema7A can affect the number of macrophages and immune cells in atherosclerotic plaques and immunity in hyperlipidemia mice. This study provides a theoretical basis for further exploring the potential of Sema7A in the prevention and treatment of atherosclerotic diseases.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R543.5
本文编号：2217159
[Abstract]:Atherosclerosis is a major cause of cardiovascular disease. In recent years, atherosclerosis is considered to be a systemic immune inflammatory disease, congenital immunity and acquired immunity are involved in it. Cellular and endothelial dysfunction. Subsequent plaque formation after injury continues to grow, eventually protruding into the lumen or rupture. When lipid plaque rupture, the formation of arterial thrombosis can lead to acute coronary syndrome (ACS), myocardial infarction or stroke and other malignant clinical events, great harm to human health. But the pathogenesis is still not very serious. The Semaphrin family is the first group of protein molecules to be found to be involved in axonal targeting. These proteins are closely related to organogenesis, angiogenesis and immune cell regulation. The Semaphrin family consists of more than 20 members and is divided into eight subgroups. Transmembrane glycosylated phosphatidyl alcohols belong to the seventh class of axon-directing molecules. Sema7A binds to beta-1 integrin to mediate olfactory nerve growth [1] and activate T-cell-mediated immune responses. Sema7A can stimulate macrophages and monocytes to produce such as IL-8, IL-6, IL-1 beta by binding to alpha-1 beta. Several recent studies have found that Sema7A plays an inflammatory role in liver fibrosis, nerve injury, pulmonary fibrosis, skin inflammation, corneal inflammation and a variety of sclerosis. Sema7A and its receptors are expressed in T lymphocytes, monocytes, dendritic cells and platelets, which are closely related to the development of atherosclerosis. This suggests that Sema7A may also mediate the migration and infiltration of inflammatory cells into the intimal submucosa during atherosclerosis. AIM: To investigate whether axon-directing molecule Sema7A can affect atherosclerotic lipid plaque formation and ultimately affect the development of atherosclerosis. Expression in the development of atherosclerosis. 2. Preparation of Sema7A-/-Apo E-/-double knockout mice. 3. Establishment of atherosclerotic mice model by feeding apo E-/-mice with high-fat diet (HFD). O-E-/-single knockout mice were fed with high-fat diet for 12 weeks before and after the body weight comparison, the presence of Sema7A or not on the weight of atherosclerotic mice were detected. The venous blood of hyperlipidemic mice was extracted from the retro-orbital venous plexus, and the serum was separated. Sema7A-/-Apo E-/-double knockout mice and Sema7A+/+Apo E-/-single knockout mice were compared. The effect of Sema7A on blood lipid in hyperlipidemic mice was analyzed. 5. Apo E-/- mice were able to form stable atherosclerotic plaques 12 weeks after feeding HFD. The whole aorta of mice was obtained by blunt isolation. Sema7A-/-Apo E-/-double knockout mice and Sema7A+/+Apo E-/-single knockout mice were compared by Sudan red staining. The distribution and size of plaque in knockout mice were studied to determine whether Sema7A affected the formation of lipid plaque in coronal and thoracic aortas of Apo E-/- mice. By extracting tissue RNA, reverse transcription and quantitative PCR, the expression of cytokines in the activated subtypes of macrophages was detected, and then the polarization of macrophages was analyzed. 7. After the establishment of atherosclerosis model, the whole blood serum was isolated and compared with that of Sema7A-/-Apo E-/-double knockout mice and Sema7A+/+Apo E-/-single knockout mice. Results: 1. The expression of Sema7A in the aorta of Apo E-/-mice changed regularly during feeding HFD. Compared with normal diet, HFD significantly increased S in aorta tissue. Sema7A-/-Apo E-/-double knockout mice were successfully prepared. 3. After 12 weeks of HFD, a large number of lipid plaques were produced in the coronal and thoracic aorta of the mice. The plaque size of the Sema7A-/-Apo E-/-double knockout mice was reduced by 51.18% (S 7 A+/+Apo E-/-single knockout mice) compared with the Sema7A-/+Apo E-/-single knockout mice. EM7A-/-Apo E-/-:5.01+1.04%, Sema7A+/+Apo E-/-:10.26+1.15%, P=0.0024).4. After the establishment of atherosclerotic mice model, compared with Sema7A+/+Apo E-/-single knockout mice, Sema7A-/-Apo E-/-double knockout mice plaque macrophages and immune T cells, dendritic cells content decreased significantly.5. Compared with Apo E-/-Sema7A+/+ single knockout mice, apo E-/-Sema7A-/-double knockout mice showed significantly increased collagen content in plaques (Sema7A-/-Apo E-/-:88.37+0.75%; Sema7A+/+Apo E-/-:82.00+1.38%, P 0.0022), -/-+/+, and the vulnerability coefficient of plaques was significantly reduced and the stability of plaques was significantly enhanced. After the establishment of atherosclerotic mice model, compared with - Sema7A +/+Apo E-/- single knockout mice, Sema7A-/-Apo E-/- double knockout mice aortic tissue type 1 macrophage M1 significantly decreased, type 2 macrophage content has a certain tendency to increase. 7. Through atherosclerotic mice plasma lipid content and immune cells related. We found that the levels of inflammatory factors were significantly lower and the expression of pro-inflammatory T cells in plasma was significantly lower in double knock mice than in single knock mice. Conclusion: Sema7A can affect the number of macrophages and immune cells in atherosclerotic plaques and immunity in hyperlipidemia mice. This study provides a theoretical basis for further exploring the potential of Sema7A in the prevention and treatment of atherosclerotic diseases.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R543.5
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1 胡淑鸿;轴突导向分子Sema7A参与小鼠动脉粥样硬化发生发展机制的研究[D];苏州大学;2015年
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