纳米粒子介导的血管紧张素Ⅱ的1型受体相关蛋白对大鼠血管内膜增生的影响

发布时间：2018-03-08 03:00

本文选题：肾素-血管紧张素-醛固酮系统　切入点：血管紧张素1型受体相关蛋白　出处：《郑州大学》2014年硕士论文　论文类型：学位论文

【摘要】：背景和目的近十余年，我国城镇人口由于生活习惯及饮食结构等因素的改变，动脉粥样硬化的发病率明显增加，其引起的心脑血管疾病已逐步上升为国人死亡的主要原因。虽然血管搭桥术及经皮血管成型术等取得了令人嘱目的成就，但是术后血管及吻合口的内膜增生，使远期通畅率常不能令人满意，使患者不能受益于这些治疗。基因治疗被视为未来从根本上解决血管病变的方法之一，高效、安全的载体系统是基因治疗的基本条件之一。纳米载体体积极小，穿透性高，甚至可以穿透整个血管壁；纳米载体多由可生物降解的有机材料制备，生物相容性好，无毒或低毒；纳米颗粒的包裹对基因有很好的保护作用。聚乳酸聚乙醇酸共聚物（PLGA）既是一种已被美国食品及药品管理局（FDA）批准的可生物降解的纳米材料，国际上已将其成功用于基因转染的研究。肾素-血管紧张素-醛固酮系统调节水盐代谢、血管张力和交感神经系统活性，血管紧张素II是其中最主要的效应肽，是血管病理生理学的重要介质。AngII必需通过其受体发挥作用，主要分布于血管平滑肌细胞（VSMC）上的I型AngII受体（AT1R），介导AngII的绝大多数生物学作用。所以AT1R基因理所当然成了动脉粥样硬化研究的重要侯选基因。近年来，Dzau, V.J在哈佛大学的实验室首先分离克隆出的AngII的1型受体相关蛋白即是一种重要的极具前景的靶基因。本实验拟于在大鼠血管损伤模型及大鼠离体VSMC上，通过纳米载体介导的基因转染的方法，评价纳米粒子携带ATRAP基因质粒转染的效率及可行性。论证纳米粒子介导的ATRAP在VSMC凋亡及抑制血管内膜增生的作用及其细胞分子机制。材料和方法本实验应用聚乳酸聚乙醇酸共聚物（PLGA）和聚乙烯醇(PVA)包载ATRAP基因质粒，制备纳米级粒子混合物，通过纳米粒子介导的基因转染的方法，将其转染至大鼠离体VSMC及大鼠颈血管损伤模型上。方法：1、纳米粒子-ATRAP基因转染体系的构建：PLGA和PVA包载ATRAP基因质粒，检测并进一步改善其粒度及体内外转染率。2、体外部分：利用纳米粒子介导的ATRAP转染的VSMC，，观察ATRAP的细胞毒性及其在体外对VSMC增殖的抑制作用和对凋亡的促进作用。3、体内部分：在被气囊导管损伤后的大鼠颈动脉局部转染纳米粒子介导的ATRAP，论证其在体内对VSMC增殖的抑制作用和凋亡的促进作用。结果 1、PLGA介导的ATRAP有较好的稳定性，释放时间约为10-14天，无明显细胞毒性。 2、大鼠血管内膜及中膜可以观察到纳米粒子介导的ATRAP的mRNA及其蛋白产物的表达。 3、在AngII刺激VSMCs48小时后，ATRAP过表达可以抑制AngII诱导的3H胸腺嘧啶核苷的合成。 4、ATRAP的过表达使磷酸化ERK的活性显著降低并明显抑制了损伤动脉的新生内膜增生。 5、ATRAP对于血管平滑肌细胞增殖的抑制作用是通过AT1受体介导ERK的激活实现的。结论 1、AT1受体介导的ERK活性在AngII调节的VSMCs增殖中起重要作用。 2、实验证明ATRAP明显抑制了AT1R介导的VSMC的增生及血管炎症反应。 3、被pcDNA3载体介导的ATRAP可以抑制VSMC的增殖及血管内膜增生。
[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.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943

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