t-PA对ApoE基因敲除小鼠血管外膜损伤致内膜增生性病变的干预作用

发布时间：2018-03-30 23:13

本文选题：组织型纤溶酶原激活物　切入点：ApoE~(-/-)小鼠　出处：《江苏大学》2017年硕士论文

【摘要】：背景和目的:动脉粥样硬化(atherosclerosis,AS)是一种动脉内壁形成由胆固醇或其他脂质,细胞废物,钙以及纤维等组成的粥样斑块,导致动脉僵硬狭窄的疾病状态,是心脑血管疾病的主要病理基础,成为危害人类健康的头号杀手。目前研究表明,遗传,免疫,炎症损伤,感染都参与了 AS的发生发展,而自主神经功能紊乱与血管内皮和免疫功能失衡密切相关,成为AS的危险因素。有学者发现机体自主神经功能紊乱早于血管病变且紊乱程度与疾病的严重程度正相关。而研究表明血管病变也同时伴有局部神经化的改变,但其具体机制目前尚仍未阐明。目前的基础研究表明,血管内膜损伤可导致动脉粥样硬化已经得到证实,有趣的是,近期研究表明血管外膜病变同样可导致动脉粥样硬化而且时间还.早于内膜病变。而且研究提示:若早期对外膜实施一定的干预措施或可能延缓或终止AS病变的进程,因此近年来对AS疾病的基础研究不仅仅局限于血管内膜和中膜,也逐渐开始关注到血管外膜的作用。Barker在颈动脉外膜剥离的动物模型的中发现血管外膜剥离后可出现相应的内膜增生性病灶。此外,免疫组化的基础研究表明:分布在动脉外膜区域的主要为去甲肾上腺素能纤维,故此我们推测外膜损伤后对交感神经的影响在动脉粥样硬化病变发生发展过程中可能起着重要作用。外周交感神经系统可合成并分泌组织型纤溶酶原激活物(tissue-type plasminogen activator,t-PA)。在生理条件下,t-PA在循环血液中也有一定水平。当血管外膜损伤时,神经纤维受到破坏,外周交感神经系统受损,将会影响t-PA的合成与释放。因此本研究在借助ApoE基因敲除小鼠建立颈动脉外膜剥离模型的基础上,以t-PA为研究对象,观察其在血管外膜损伤致内膜增生性病变中的作用。本实验我们以ApoE基因敲除小鼠为研究对象,随机分成三组:对照组、外膜损伤组、t-PA组(外膜损伤组+静脉注射t-PA:156μg/day);探讨以小鼠建立颈动脉外膜剥离模型的可行性,并观察外源性给予t-PA对交感神经、血管损伤后内膜增生的影响。采用苏木素-伊红染色(HE染色)观察ApoE基因敲除小鼠动脉粥样硬化病变情况,免疫组织化学染色方法检测血管局部病变成分,免疫荧光染色方法及western blot检测络氨酸轻化酶(tyrosine hydroxylase,TH)的表达情况。结果:(1)胶原酶消化+显微镊分离小鼠血管外膜的方法,可有效地破坏血管外膜结构的完整性,达到血管外膜损伤的效果。(2)外膜剥离2周后可见相应内膜处出现增生性病变,t-PA组可见明显增生性病变,内膜病变面积较外膜损伤组明显变大,其差异有统计学意义(P0.05)。(3)SM-α-actin免疫组化结果显示:对照组中仅有中膜处有淡黄色物质表达,而外膜和内膜并无黄色物质出现;外膜损伤组、t-PA组血管内膜及中膜有大量均匀阳性着色,其主要病变成分是平滑肌细胞,但是t-PA组病变较外膜损伤组明显增强。(4)免疫荧光结果显示:t-PA组与外膜损伤组比较,TH荧光亮度增强;外膜损伤组与对照组相比,TH荧光强度增强。(5)Western blot结果显示:外膜损伤组较对照组,TH蛋白表达量增强,但较t-PA组TH蛋白表达量减弱。结论:(1)ApoE基因敲除小鼠颈动脉外膜剥离后能有效地建立早期动脉粥样硬化的模型。(2)t-PA干预后可增加外膜损伤后斑块面积及促进交感神经递质的释放,促进动脉粥样硬化的进展。
[Abstract]:Background and objective: atherosclerosis (atherosclerosis, AS) is a kind of arterial wall formed by cholesterol or other lipid composition, cell waste, calcium and fiber plaques, cause arterial stiffness stenosis disease state, is the main pathological basis of cardiovascular and cerebrovascular diseases, has become the first killer of human health. The present study showed that genetic the immune inflammatory injury, infection, and are involved in the occurrence and development of AS, and the dysfunction of the autonomic nervous system and vascular endothelial and immune imbalance is closely related to become the risk factors of AS. There is a positive correlation of severity scholar find body dysfunction of the autonomic nervous system in vascular disease and the degree of disorder and disease. But research shows that blood vessels at the same time with the local nerve lesions change, but the exact mechanism is not clear. The current basic research showed that vascular intimal injury can cause the artery Atherosclerosis has been confirmed, interestingly, recent studies showed that adventitial lesions can also lead to atherosclerosis and time. As early as in endometrial lesions. But research suggests that if foreign film early intervention or may delay or terminate the AS disease process, so in recent years, based on research of AS disease is not limited to the intima and media have gradually started to pay attention to the role of.Barker in the adventitia of animal model of carotid adventitial stripping found in adventitia after stripping intimal hyperplasia lesions can appear accordingly. In addition, based on immunohistochemistry showed that the distribution area is mainly in the adventitia of noradrenergic fibers. Therefore, we speculate that the process of adventitial injury effect on sympathetic nerve occurred in atherosclerotic lesions may play an important role in development of peripheral sympathetic. Nervous system synthesis and secretion of tissue type plasminogen activator (tissue-type plasminogen, activator, t-PA). Under physiological conditions, t-PA also has a certain level in the circulating blood. When the vascular adventitial injury, nerve damage, impaired peripheral sympathetic nervous system, will affect the synthesis and release of t-PA in this study. With the help of ApoE gene knockout mice to establish carotid adventitial stripping based on the model, using t-PA as the research object, observe the injury caused by intimal hyperplasia in vascular adventitia. In this experiment, we used ApoE knockout mice as the research object, randomly divided into three groups: control group, adventitial injury group, t-PA group (intravenous injection of t-PA:156 + adventitia injury group g/day); to explore the feasibility to establish mouse carotid adventitial stripping model, and the effect of exogenous t-PA on sympathetic nerve, the intimal hyperplasia after vascular injury Effect. Using hematoxylin eosin staining (HE staining) was observed in ApoE knockout mouse atherosclerotic lesions, immunohistochemistry methods of vascular lesions, immunofluorescence staining method and Western blot detection of tyrosine hydroxylase (tyrosine hydroxylase, TH) expression. Results: (1) method collagenase digestion + microforceps isolated from mouse vascular adventitia, which can effectively destroy the integrity of the adventitia structure, to achieve the effect of adventitia injury. (2) the outer membrane peeling after 2 weeks at the corresponding endometrial hyperplastic lesions, t-PA group showed obvious hyperplasia, with outer membrane injury group endometrial lesions area became larger., the difference was statistically significant (P0.05). (3) SM- alpha -actin immunohistochemistry results showed that: in the control group only in the membrane expression of pale yellow substance, and the outer and inner no yellow substance; membrane damage Injury group, t-PA group intimal many uniform positive staining, its main ingredient is lesions of smooth muscle cells, but the pathological changes in group t-PA was significantly enhanced. The adventitia injury group (4) immunofluorescence results showed that: t-PA group compared with the adventitia injury group, TH fluorescent brightness enhancement; outer membrane injury group compared with the control group and enhance the fluorescence intensity of TH (5). Western blot showed that adventitial injury group than in the control group, the expression of TH protein increased, but compared with t-PA group, the expression of TH protein decreased. Conclusion: (1) ApoE knockout mice carotid artery adventitia after stripping can effectively establish a model of early atherosclerosis (2). After t-PA intervention can increase the adventitial injury area of plaque and promote the sympathetic neurotransmitter release, promote the progression of atherosclerosis.

【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R543.5

【参考文献】