失活氨基脲敏感性胺氧化酶对动脉粥样硬化发展的影响

发布时间：2018-03-26 04:54

本文选题：氨基脲敏感性胺氧化酶　切入点：动脉粥样硬化　出处：《苏州大学》2016年硕士论文

【摘要】：目的:氨基脲敏感性胺氧化酶(semicarbazide-sensitive amine oxidase,SSAO)是一种具有双功能性的分子,粘附作用和氧化脱氨作用,两个作用都与酶的活力紧密相关。临床研究发现血清中SSAO的活性与颈动脉粥样硬化斑块的大小呈正相关性,但是SSAO在动脉粥样硬化发展中的作用尚不清楚。本探究旨在探讨失活SSAO对动脉粥样硬化发展的影响及其机制。方法:a)预防给药实验:给予雌性LDLr knockout(LDLrKO)小鼠高脂高胆固醇食物(Western-type diet/WTD)以促成动脉粥样硬化的发生(早期病灶)和发展(晚期病灶)。期间将氨基脲(semicarbazide,SCZ)(0.125%)添加到小鼠饮用水中来抑制SSAO在小鼠体内的活性;b)治疗给药实验:雌性和雄性LDLrKO小鼠通过喂食WTD诱导动脉粥样硬化斑块的形成。随后,SCZ(0.125%)被添加到小鼠饮用水中。雌性小鼠依旧喂食WTD并给予SCZ,而雄性小鼠则换成不含胆固醇的食物(chow diet)以降低血脂并给予SCZ。小鼠解剖后油红染色检测其病灶大小;免疫组织化学技术检测病灶成分;流式细胞术检测炎细胞浸润以及主动脉平滑肌细胞(smooth muscle cells,SMCs)的增殖状态;划痕实验检测体外平滑肌细胞迁移能力。结果:a)预防给药实验结果:SCZ可完全抑制体内SSAO的活性,但对其蛋白的表达水平并无影响;尽管血浆中的总胆固醇水平没有变化,利用SCZ诱导SSAO失活不仅使早期(1.5-fold,P=0.0043)和晚期(1.8-fold,P=0.0013)动脉粥样硬化病灶增大,而且导致斑块中巨噬细胞的含量降低而SMCs的含量升高(巨噬细胞:~0.74-fold,P=0.0002(早期)/0.0016(晚期);SMCs:~1.55-fold,P=0.0003(早期)/0.0001(晚期));SSAO失活抑制循环中的单核细胞向外周组织浸润并且显著地降低了循环中的Ly6C高表达单核细胞的数量(0.7-fold,P=0.0001),这一点有可能是病灶中巨噬细胞减少的原因,而饮用SCZ小鼠体内斑块中SMCs的增多则可归因于SMCs表型的改变,这一点也被病灶中增殖的SMCs和胶原含量增加及体外SMCs培养实验结果所证实。b)治疗给药实验结果:尽管血浆胆固醇水平和病灶大小都没有发生变化,SSAO失活都能通过增加胶原蛋白含量(高血脂:1.5-fold,P0.05;降脂后:1.9-fold,P0.05)增加病灶的稳定性,这一作用与血脂的高低无关;在高血脂状态下,SSAO失活还导致血液中Ly6C高表达的单核细胞(0.32-fold,P0.001)和病灶中巨噬细胞(0.70-fold,P0.05)的减少,但在降脂实验小鼠中并未发现这些改变;值得注意的是,SSAO失活除增加了病灶中SMCs的数量(高血脂:1.4-fold,P0.05;降脂后:1.7-fold,P0.05),其中合成表型(Ki67+)的SMCs数量也大幅度增长(高血脂:1.5-fold,P0.05;降脂后:3.3-fold,P0.05)。结论:SCZ诱导SSAO失活可促进SMCs向合成表型转变。这种表型的改变尽管有可能促进斑块的增大,却可显著提升斑块的稳定性。因此,对于SSAO活性的调节也许可以成为治疗动脉粥样硬化的新方法。
[Abstract]:Objective: semicarbazide-sensitive amine oxidase (SSAO) is a bifunctional molecule with adhesion and oxidative deamination. Clinical studies showed that the activity of SSAO in serum was positively correlated with the size of carotid atherosclerotic plaque. However, the role of SSAO in the development of atherosclerosis is not clear. This study aims to explore the effects of inactivated SSAO on atherosclerosis and its mechanism. Methods: preventive administration of SSAO: female LDLr knockout LDLrKOs with hyperlipidemia and gallbladder. Steroid diet Western-type dietr WTD to promote the occurrence and development of atherosclerosis (early lesion) and progression (during which semicarbazide was added to the drinking water of mice to inhibit the activity of SSAO in mice). Sexual and male LDLrKO mice were fed WTD to induce atherosclerotic plaque formation. Then SCZ 0.125 was added to the drinking water of the mice. The female mice were still fed WTD and given SCZs, while the male mice were replaced with cholesterol free food. Dietate was used to reduce blood lipids and the lesion size was detected by oil red staining after dissection of SCZ. mice. The inflammatory cell infiltration and smooth muscle cells (SMC) proliferation of aortic smooth muscle cells were detected by immunohistochemistry and flow cytometry. The results of the scratch test were used to detect the migration ability of smooth muscle cells in vitro. Results: the prophylactic administration experiment showed that the activity of SSAO in vivo was inhibited completely, but the protein expression level was not affected, although the total cholesterol level in plasma did not change. Inactivation of SSAO induced by SCZ not only increased the atherosclerotic lesions in the early stage (1.5-foldd) and in the late stage (1.8-foldd P _ (0.0013)), but also increased the number of atherosclerotic lesions. It also resulted in a decrease in macrophage content in plaque and an increase in SMCs content (macrophage: 0.74-foldP#en0# 0.0002) (early / late / 0. 0016 / 0. 002 / 0. 0003 / 0. 0003 / 0. 0003 / 0. 0001 / 0. 0001 / 0. 0001 inactivation of mononuclear cells in the circulation inhibits the infiltration of mononuclear cells into peripheral tissues and significantly reduces the circulation. The number of mononuclear cells expressed in Ly6C is 0.7-foldP0.0001, which may be the reason for the decrease of macrophages in the lesions. The increase of SMCs in plaque of SCZ mice was attributed to the change of SMCs phenotype. This is also confirmed by the increase in proliferative SMCs and collagen content in the lesion and by the results of SMCs culture in vitro. Increased collagen content (hyperlipidemia, 1. 5-foldd, P0. 05; after lipid lowering, 1. 9-foldP0. 05) increased the stability of the lesion, This effect was not related to the level of blood lipids, but the inactivation of SSAO in hyperlipidemia also resulted in the decrease of monocytes with high expression of Ly6C and macrophages (0.70 -folddP0.05) in blood, but these changes were not found in lipid-lowering mice. It is worth noting that the inactivation of SSAO increases the number of SMCs in the lesion (hyperlipidemia: 1.4-foldP0.05; after lowering fat, 1.7-fold P0.05, in which the synthetic phenotype is P0.05), and the number of SMCs also increases significantly (hyperlipidemia: 1.5-foldtad: P0.05; after lipid-lowering, w 3.3-foldd; P0.05. conclusion: SSAO inactivation can promote SMCs alignment. Phenotypic changes. This phenotypic change, though likely to promote plaque enlargement, Therefore, the regulation of SSAO activity may be a new method for the treatment of atherosclerosis.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R543.5

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