軸突導(dǎo)向分子Sema7A參與小鼠動脈粥樣硬化發(fā)生發(fā)展機制的研究
發(fā)布時間：2018-09-01 12:08
【摘要】：動脈粥樣硬化是引發(fā)心血管疾病的主要原因。近年來,動脈粥樣硬化被認(rèn)為是一種系統(tǒng)性免疫炎癥的疾病,先天性免疫與獲得性免疫均參與其。損傷早期形態(tài)學(xué)變化即脂質(zhì)條紋,是一種嚴(yán)重的代謝和免疫損傷,表現(xiàn)為血管張力異常,炎癥,細胞和內(nèi)皮功能障礙。隨后損傷形成斑塊繼續(xù)生長,最終凸入管腔或發(fā)生破裂。當(dāng)脂質(zhì)斑塊破裂后,形成的動脈血栓會導(dǎo)致急性冠脈綜合征(ACS)、心肌梗死或腦中風(fēng)等惡性臨床事件,對人類的健康危害極大。但其發(fā)病機制目前仍不是十分清楚,尋找有效藥物對該疾病的治療迫在眉睫。Semaphrin家族是最初以參與軸突導(dǎo)向發(fā)現(xiàn)的一類蛋白分子家族,該類蛋白與組織器官的形成,血管生成與免疫細胞調(diào)控密切相關(guān)。Semaphrin家族包括20多個成員共分成8個亞類,Semaphrin7A(Sema7A)是跨膜的糖基化磷脂�；嫉鞍�,屬于第七類軸突導(dǎo)向分子家族,Sema7A能夠與β1整合素結(jié)合從而介導(dǎo)嗅覺神經(jīng)的生長[1]并啟動激活由T細胞介導(dǎo)的免疫應(yīng)答。Sema7A蛋白可以通過與α1β1結(jié)合刺激巨噬細胞和單核細胞產(chǎn)生諸如IL-8,IL-6,IL-1β以及TNF-α等炎癥因子。近期另有多篇研究發(fā)現(xiàn),Sema7A在肝纖維化,神經(jīng)損傷,肺纖維化,皮膚炎癥,角膜炎癥和多種硬化癥中均有發(fā)揮促炎作用。盡管目前我們得知Sema7A參與多種生理性過程及病理性疾病中,但Sema7A是否參與動脈粥樣硬化疾病的發(fā)生尚無報道。Sema7A及其受體在T淋巴細胞、單核細胞,樹突狀細胞以及血小板中均有表達,而這些細胞恰恰是與動脈粥樣硬化的發(fā)生有著密切關(guān)系的細胞種類。且有文獻證明內(nèi)皮細胞上的Sema7A可以誘導(dǎo)缺氧過程中中性粒細胞向內(nèi)皮下層的遷移浸潤過程,這也提示Sema7A或在動脈粥樣硬化過程中也可以介導(dǎo)炎癥細胞向內(nèi)膜下層的遷移浸潤。因此我們著手研究Sema7A在動脈粥樣硬化過程中的作用,并發(fā)現(xiàn)Sema7A敲除的情況下可以減少載脂蛋白E敲除小鼠的斑塊形成并減緩動脈粥樣硬化的發(fā)展。目的:本文旨在探討軸突導(dǎo)向分子Sema7A是否能夠影響動脈粥樣硬化脂質(zhì)斑塊形成,最終影響動脈粥樣硬化的發(fā)展。研究方法:1.利用Apo E-/-小鼠喂食高脂飼料1周,4周,12周,通過RNA以及蛋白水平檢測Sema7A及受體在動脈粥樣硬化發(fā)展過程中的表達情況。2.制備Sema7A-/-Apo E-/-雙敲除小鼠。3.通過對載脂蛋白E(Apo E)敲除小鼠(以下簡寫為Apo E-/-小鼠)喂食高脂飼料(high-fat diet,以下簡寫為HFD),建立動脈粥樣硬化小鼠模型。4.Sema7A-/-Apo E-/-雙敲除小鼠與Sema7A+/+Apo E-/-單敲除小鼠,,在喂食高脂飼料12周前后的體重對比,檢測Sema7A存在與否對動脈粥樣硬化小鼠的體重產(chǎn)生影響。并通過眼眶后靜脈叢抽取高脂血癥小鼠的靜脈血,分離血清,檢測比較Sema7A-/-Apo E-/-雙敲除小鼠與Sema7A+/+Apo E-/-單敲除小鼠血漿中的血脂含量,分析Sema7A對高脂血癥狀態(tài)下小鼠血脂含量的影響。5.當(dāng)喂食HFD 12周后,Apo E-/-小鼠能夠形成穩(wěn)定的動脈粥樣硬化斑塊,鈍性分離得到小鼠整只主動脈,通過蘇丹紅染色,對比Sema7A-/-Apo E-/-雙敲除小鼠與Sema7A+/+Apo E-/-單敲除小鼠斑塊的分布與大小,研究Sema7A是否影響Apo E-/-小鼠冠狀及胸主動脈中脂質(zhì)斑塊的形成。并通過對比兩組小鼠斑塊內(nèi)細胞種類以及分布的不同,研究Sema7A存在與對斑塊內(nèi)細胞成分的影響。6.取形成穩(wěn)定斑塊的小鼠的主動脈組織,通過提取組織RNA,逆轉(zhuǎn)錄并通過定量PCR方法檢測巨噬細胞活化亞型的相應(yīng)細胞因子表達,進而分析巨噬細胞的極化情況。7.動脈粥樣硬化模型構(gòu)建成功后,分離全血血清,比較Sema7A-/-Apo E-/-雙敲除小鼠與Sema7A+/+Apo E-/-單敲除小鼠血漿中T細胞活化分泌的細胞因子的水平以及炎癥因子的水平,檢測Sema7A對高脂血癥狀態(tài)下小鼠炎癥因子含量的影響。結(jié)果:1.在喂食HFD期間,Apo E-/-小鼠主動脈內(nèi)Sema7A的表達呈規(guī)律變化。與喂食普通飼料相比,喂食HFD可以明顯上調(diào)主動脈組織中Sema7A的表達,并且在喂食一周時Sema7A的表達量達到最高。2.成功制備了Sema7A-/-Apo E-/-雙敲除小鼠。3.小鼠在喂食12周HFD后其冠狀及胸主動脈中產(chǎn)生了大量脂質(zhì)斑塊,Sema7A-/-Apo E-/-雙敲除小鼠斑塊大小較Sema7A+/+Apo E-/-單敲除小鼠降低了51.18%(Sema7A-/-Apo E-/-:5.01±1.04%,Sema7A+/+Apo E-/-:10.26±1.15%,P=0.0024)。4.動脈粥樣硬化小鼠模型建立后,與Sema7A+/+Apo E-/-單敲除小鼠相比,Sema7A-/-Apo E-/-雙敲除小鼠的斑塊內(nèi)巨噬細胞以及免疫細胞T細胞,樹突狀細胞的含量有比較明顯的降低。5.通過主動脈根部斑塊切片染色發(fā)現(xiàn),與Apo E-/-Sema7A+/+單敲除小鼠相比,Apo E-/-Sema7A-/-雙敲除小鼠的斑塊內(nèi)膠原含量明顯上調(diào)(Sema7A-/-Apo E-/-:88.37±0.75%;Sema7A+/+Apo E-/-:82.00±1.38%,P0.0022),-/-+/+,且斑塊的易破損系數(shù)明顯降低,斑塊的穩(wěn)定性明顯增強。6.動脈粥樣硬化小鼠模型建立后,與-Sema7A+/+Apo E-/-單敲除小鼠相比,Sema7A-/-Apo E-/-雙敲除小鼠主動脈組織中1型巨噬細胞M1有明顯的降低,2型巨噬細胞的含量有一定的升高趨勢。7.通過動脈粥樣硬化小鼠的血漿中血脂含量以及免疫細胞相關(guān)炎癥因子的檢測,我們發(fā)現(xiàn),相較于單敲小鼠,雙敲除小鼠的炎癥因子的水平明顯降低且血漿中促炎T細胞表達明顯下調(diào)。結(jié)論:我們闡明了Sema7A通過影響高脂血癥狀態(tài)下小鼠動脈粥樣硬化斑塊內(nèi)巨噬細胞以及免疫細胞的數(shù)量以及免疫應(yīng)答進而影響到主動脈脂質(zhì)斑塊的形成,從而抑制了動脈粥樣硬化的發(fā)展這一機制。該研究為利用Sema7A進一步開發(fā)研究預(yù)防和治療動脈粥樣硬化疾病的潛力提供了理論基礎(chǔ)。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R543.5
本文編號：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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R543.5
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1 胡淑鴻;軸突導(dǎo)向分子Sema7A參與小鼠動脈粥樣硬化發(fā)生發(fā)展機制的研究[D];蘇州大學(xué);2015年
，本文編號：2217159
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