本文選題：腎素-血管緊張素-醛固酮系統(tǒng)　切入點(diǎn)：血管緊張素1型受體相關(guān)蛋白　出處：《鄭州大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：背景和目的 近十余年，我國城鎮(zhèn)人口由于生活習(xí)慣及飲食結(jié)構(gòu)等因素的改變，動脈粥樣硬化的發(fā)病率明顯增加，其引起的心腦血管疾病已逐步上升為國人死亡的主要原因。雖然血管搭橋術(shù)及經(jīng)皮血管成型術(shù)等取得了令人囑目的成就，但是術(shù)后血管及吻合口的內(nèi)膜增生，使遠(yuǎn)期通暢率常不能令人滿意，使患者不能受益于這些治療。 基因治療被視為未來從根本上解決血管病變的方法之一，高效、安全的載體系統(tǒng)是基因治療的基本條件之一。納米載體體積極小，穿透性高，甚至可以穿透整個血管壁；納米載體多由可生物降解的有機(jī)材料制備，生物相容性好，無毒或低毒；納米顆粒的包裹對基因有很好的保護(hù)作用。聚乳酸聚乙醇酸共聚物（PLGA）既是一種已被美國食品及藥品管理局（FDA）批準(zhǔn)的可生物降解的納米材料，國際上已將其成功用于基因轉(zhuǎn)染的研究。 腎素-血管緊張素-醛固酮系統(tǒng)調(diào)節(jié)水鹽代謝、血管張力和交感神經(jīng)系統(tǒng)活性，血管緊張素II是其中最主要的效應(yīng)肽，是血管病理生理學(xué)的重要介質(zhì)。AngII必需通過其受體發(fā)揮作用，主要分布于血管平滑肌細(xì)胞（VSMC）上的I型AngII受體（AT1R），介導(dǎo)AngII的絕大多數(shù)生物學(xué)作用。所以AT1R基因理所當(dāng)然成了動脈粥樣硬化研究的重要侯選基因。近年來，Dzau, V.J在哈佛大學(xué)的實(shí)驗(yàn)室首先分離克隆出的AngII的1型受體相關(guān)蛋白即是一種重要的極具前景的靶基因。 本實(shí)驗(yàn)擬于在大鼠血管損傷模型及大鼠離體VSMC上，通過納米載體介導(dǎo)的基因轉(zhuǎn)染的方法，評價納米粒子攜帶ATRAP基因質(zhì)粒轉(zhuǎn)染的效率及可行性。論證納米粒子介導(dǎo)的ATRAP在VSMC凋亡及抑制血管內(nèi)膜增生的作用及其細(xì)胞分子機(jī)制。 材料和方法 本實(shí)驗(yàn)應(yīng)用聚乳酸聚乙醇酸共聚物（PLGA）和聚乙烯醇(PVA)包載ATRAP基因質(zhì)粒，制備納米級粒子混合物，通過納米粒子介導(dǎo)的基因轉(zhuǎn)染的方法，將其轉(zhuǎn)染至大鼠離體VSMC及大鼠頸血管損傷模型上。 方法：1、納米粒子-ATRAP基因轉(zhuǎn)染體系的構(gòu)建：PLGA和PVA包載ATRAP基因質(zhì)粒，檢測并進(jìn)一步改善其粒度及體內(nèi)外轉(zhuǎn)染率。2、體外部分：利用納米粒子介導(dǎo)的ATRAP轉(zhuǎn)染的VSMC，，觀察ATRAP的細(xì)胞毒性及其在體外對VSMC增殖的抑制作用和對凋亡的促進(jìn)作用。3、體內(nèi)部分：在被氣囊導(dǎo)管損傷后的大鼠頸動脈局部轉(zhuǎn)染納米粒子介導(dǎo)的ATRAP，論證其在體內(nèi)對VSMC增殖的抑制作用和凋亡的促進(jìn)作用。 結(jié)果 1、PLGA介導(dǎo)的ATRAP有較好的穩(wěn)定性，釋放時間約為10-14天，無明顯細(xì)胞毒性。 2、大鼠血管內(nèi)膜及中膜可以觀察到納米粒子介導(dǎo)的ATRAP的mRNA及其蛋白產(chǎn)物的表達(dá)。 3、在AngII刺激VSMCs48小時后，ATRAP過表達(dá)可以抑制AngII誘導(dǎo)的3H胸腺嘧啶核苷的合成。 4、ATRAP的過表達(dá)使磷酸化ERK的活性顯著降低并明顯抑制了損傷動脈的新生內(nèi)膜增生。 5、ATRAP對于血管平滑肌細(xì)胞增殖的抑制作用是通過AT1受體介導(dǎo)ERK的激活實(shí)現(xiàn)的。 結(jié)論 1、AT1受體介導(dǎo)的ERK活性在AngII調(diào)節(jié)的VSMCs增殖中起重要作用。 2、實(shí)驗(yàn)證明ATRAP明顯抑制了AT1R介導(dǎo)的VSMC的增生及血管炎癥反應(yīng)。 3、被pcDNA3載體介導(dǎo)的ATRAP可以抑制VSMC的增殖及血管內(nèi)膜增生。
[Abstract]:Background and purpose
Over the past ten years, China's urban population due to the living habits and the dietary factors change, atherosclerosis significantly increased incidence of cardiovascular and cerebrovascular diseases caused by the gradually increased as the main reason of death. Although vascular bypass surgery and percutaneous angioplasty and has made a great achievement, but after the operation and vascular anastomotic intimal hyperplasia, the long-term patency rate is often not satisfactory, so that patients can not benefit from the treatment.
Gene therapy is regarded as one of the methods in the future, fundamentally solve the vascular lesions of efficient vector system security is one of the basic conditions for gene therapy. The nano carrier small volume, high penetration, can even penetrate the entire vascular wall; nanoparticles by biodegradable organic material preparation, good biocompatibility. Non-toxic or low toxicity; nanoparticles coated with good protective effect on the gene. Polylactic co glycolic acid (PLGA) is a kind of has been the United States Food and Drug Administration (FDA) approved biodegradable nano materials, which have been successfully used in the research of gene transfection.
The renin angiotensin aldosterone system regulates the metabolism of water and salt, vascular tension and sympathetic nervous system activity, angiotensin II is one of the main effector peptide of.AngII, are important mediators of vascular pathophysiology must play a role through its receptor, mainly in vascular smooth muscle cells (VSMC) on the I type AngII receptor (AT1R), most of the biological effects of AngII mediated AT1R gene. So behoove becomes an important candidate gene Hou atherosclerosis research. In recent years, Dzau, type 1 receptor related protein V.J in the laboratories of the Harvard University first cloned the target gene AngII is a very important prospect.
In this experiment, to be in a rat model of vascular injury and isolated rat VSMC, through the method of gene transfection mediated by the efficiency and feasibility of nanoparticles with ATRAP gene plasmid transfection. Demonstration of nanoparticle mediated ATRAP in VSMC apoptosis and inhibit intimal hyperplasia and cellular molecular mechanism.
Materials and methods
In this study, poly (lactic acid glycolic acid) copolymer (PLGA) and polyvinyl alcohol (PVA) were used to carry out ATRAP gene plasmid, and nano particle mixture was prepared. Then transfected into the rat VSMC and rat carotid artery injury model by nanoparticle mediated gene transfection.
Methods: 1 construction of -ATRAP nanoparticles gene delivery system: PLGA and PVA encapsulated ATRAP gene plasmid detection, and further improve its particle size and in vitro and in vivo transfection rate of.2 transfected by ATRAP in vitro: nanoparticle mediated cytotoxicity of VSMC, ATRAP and observe the in vitro proliferation of VSMC and inhibition of apoptosis the role of.3 in vivo: the balloon catheter injury after rat carotid artery local transfection mediated by nanoparticles of ATRAP, demonstrate the in vivo inhibition of apoptosis and proliferation of VSMC promotion.
Result
1, PLGA mediated ATRAP has good stability, and the release time is about 10-14 days without obvious cytotoxicity.
2, the expression of mRNA and its protein products of ATRAP mediated by nanoparticles can be observed in the intima and middle membrane of the rat.
3, after AngII stimulation for VSMCs48 hours, overexpression of ATRAP can inhibit the synthesis of AngII induced 3H thymidine.
4, the overexpression of ATRAP significantly reduced the activity of phosphorylated ERK and significantly inhibited the neointimal hyperplasia of the injured artery.
5, the inhibitory effect of ATRAP on the proliferation of vascular smooth muscle cells is realized by the activation of the AT1 receptor to mediate the activation of ERK.
conclusion
1, the ERK activity mediated by AT1 receptor plays an important role in the proliferation of AngII regulated VSMCs.
2, the experimental results showed that ATRAP significantly inhibited the proliferation of AT1R mediated VSMC and the vascular inflammatory response.
3, ATRAP mediated by pcDNA3 can inhibit the proliferation of VSMC and the proliferation of vascular intima.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R943

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