發(fā)布時(shí)間：2018-03-30 23:13

  本文選題：組織型纖溶酶原激活物　切入點(diǎn)：ApoE~(-/-)小鼠　出處：《江蘇大學(xué)》2017年碩士論文

【摘要】：背景和目的:動(dòng)脈粥樣硬化(atherosclerosis,AS)是一種動(dòng)脈內(nèi)壁形成由膽固醇或其他脂質(zhì),細(xì)胞廢物,鈣以及纖維等組成的粥樣斑塊,導(dǎo)致動(dòng)脈僵硬狹窄的疾病狀態(tài),是心腦血管疾病的主要病理基礎(chǔ),成為危害人類(lèi)健康的頭號(hào)殺手。目前研究表明,遺傳,免疫,炎癥損傷,感染都參與了 AS的發(fā)生發(fā)展,而自主神經(jīng)功能紊亂與血管內(nèi)皮和免疫功能失衡密切相關(guān),成為AS的危險(xiǎn)因素。有學(xué)者發(fā)現(xiàn)機(jī)體自主神經(jīng)功能紊亂早于血管病變且紊亂程度與疾病的嚴(yán)重程度正相關(guān)。而研究表明血管病變也同時(shí)伴有局部神經(jīng)化的改變,但其具體機(jī)制目前尚仍未闡明。目前的基礎(chǔ)研究表明,血管內(nèi)膜損傷可導(dǎo)致動(dòng)脈粥樣硬化已經(jīng)得到證實(shí),有趣的是,近期研究表明血管外膜病變同樣可導(dǎo)致動(dòng)脈粥樣硬化而且時(shí)間還.早于內(nèi)膜病變。而且研究提示:若早期對(duì)外膜實(shí)施一定的干預(yù)措施或可能延緩或終止AS病變的進(jìn)程,因此近年來(lái)對(duì)AS疾病的基礎(chǔ)研究不僅僅局限于血管內(nèi)膜和中膜,也逐漸開(kāi)始關(guān)注到血管外膜的作用。Barker在頸動(dòng)脈外膜剝離的動(dòng)物模型的中發(fā)現(xiàn)血管外膜剝離后可出現(xiàn)相應(yīng)的內(nèi)膜增生性病灶。此外,免疫組化的基礎(chǔ)研究表明:分布在動(dòng)脈外膜區(qū)域的主要為去甲腎上腺素能纖維,故此我們推測(cè)外膜損傷后對(duì)交感神經(jīng)的影響在動(dòng)脈粥樣硬化病變發(fā)生發(fā)展過(guò)程中可能起著重要作用。外周交感神經(jīng)系統(tǒng)可合成并分泌組織型纖溶酶原激活物(tissue-type plasminogen activator,t-PA)。在生理?xiàng)l件下,t-PA在循環(huán)血液中也有一定水平。當(dāng)血管外膜損傷時(shí),神經(jīng)纖維受到破壞,外周交感神經(jīng)系統(tǒng)受損,將會(huì)影響t-PA的合成與釋放。因此本研究在借助ApoE基因敲除小鼠建立頸動(dòng)脈外膜剝離模型的基礎(chǔ)上,以t-PA為研究對(duì)象,觀(guān)察其在血管外膜損傷致內(nèi)膜增生性病變中的作用。本實(shí)驗(yàn)我們以ApoE基因敲除小鼠為研究對(duì)象,隨機(jī)分成三組:對(duì)照組、外膜損傷組、t-PA組(外膜損傷組+靜脈注射t-PA:156μg/day);探討以小鼠建立頸動(dòng)脈外膜剝離模型的可行性,并觀(guān)察外源性給予t-PA對(duì)交感神經(jīng)、血管損傷后內(nèi)膜增生的影響。采用蘇木素-伊紅染色(HE染色)觀(guān)察ApoE基因敲除小鼠動(dòng)脈粥樣硬化病變情況,免疫組織化學(xué)染色方法檢測(cè)血管局部病變成分,免疫熒光染色方法及western blot檢測(cè)絡(luò)氨酸輕化酶(tyrosine hydroxylase,TH)的表達(dá)情況。結(jié)果:(1)膠原酶消化+顯微鑷分離小鼠血管外膜的方法,可有效地破壞血管外膜結(jié)構(gòu)的完整性,達(dá)到血管外膜損傷的效果。(2)外膜剝離2周后可見(jiàn)相應(yīng)內(nèi)膜處出現(xiàn)增生性病變,t-PA組可見(jiàn)明顯增生性病變,內(nèi)膜病變面積較外膜損傷組明顯變大,其差異有統(tǒng)計(jì)學(xué)意義(P0.05)。(3)SM-α-actin免疫組化結(jié)果顯示:對(duì)照組中僅有中膜處有淡黃色物質(zhì)表達(dá),而外膜和內(nèi)膜并無(wú)黃色物質(zhì)出現(xiàn);外膜損傷組、t-PA組血管內(nèi)膜及中膜有大量均勻陽(yáng)性著色,其主要病變成分是平滑肌細(xì)胞,但是t-PA組病變較外膜損傷組明顯增強(qiáng)。(4)免疫熒光結(jié)果顯示:t-PA組與外膜損傷組比較,TH熒光亮度增強(qiáng);外膜損傷組與對(duì)照組相比,TH熒光強(qiáng)度增強(qiáng)。(5)Western blot結(jié)果顯示:外膜損傷組較對(duì)照組,TH蛋白表達(dá)量增強(qiáng),但較t-PA組TH蛋白表達(dá)量減弱。結(jié)論:(1)ApoE基因敲除小鼠頸動(dòng)脈外膜剝離后能有效地建立早期動(dòng)脈粥樣硬化的模型。(2)t-PA干預(yù)后可增加外膜損傷后斑塊面積及促進(jìn)交感神經(jīng)遞質(zhì)的釋放,促進(jìn)動(dòng)脈粥樣硬化的進(jìn)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.

【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R543.5

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本文編號(hào)：1688081