长链非编码RNA-MIAT在眼部神经和血管功能异常过程的调控作用研究

发布时间：2018-02-04 07:29

本文关键词： 长链非编码RNA 血管生成神经退行性病变反应性神经胶质细胞　出处：《南京医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:血管和神经系统虽然在功能上有所不同,但它们通常具有类似的功能维护机制以及相同的调控因子。神经血管功能紊乱已成为一些血管性疾病和神经性疾病的发病机制。本研究旨在阐明长链非编码RNA-MIAT(long non-coding RNA-MIAT)在眼部神经血管功能异常进程的调控作用及其机制。方法:我们首先通过qRT-PCR检测了 MIAT在视网膜血管病变和神经病变动物模型以及视网膜血管细胞和神经细胞病变中的表达规律,明确了眼部血管神经病变与MIAT表达的相关性;在体水平,采用视网膜血管发育、角膜新生血管(CNV)以及糖尿病视网膜病变(DR)等动物模型,通过视网膜平铺片IB4染色、裂隙灯显微镜和免疫荧光染色等技术,揭示了 MIAT干预对眼部神经血管功能异常的调控作用;离体水平,采用Hoechst染色、JC-1染色和PI/Calcein-AM染色等技术,揭示了 MIAT表达干预对视网膜细胞活力、增殖、凋亡和细胞相互作用的影响;机制研究方面,采用荧光素酶和qRT-PCR等实验,揭示了 MIAT调控Muller细胞功能的分子机制。结果:qRT-PCR检测发现,MIAT在氧诱导视网膜病变(OIR)、视神经夹伤(ONT)以及糖尿病的动物模型中表达异常;MIAT在Miiller细胞、内皮细胞、周细胞以及神经元的高氧或者低氧胁迫时表达异常,其中MIAT在Miiller细胞胁迫时表达改变最为明显。在体实验发现,MIAT的表达沉默抑制了小鼠视网膜血管的发育,缓解了角膜碱烧伤小鼠角膜新生血管的形成,减少了糖尿病小鼠视网膜的胶质化以及RGC的存活。离体实验发现,MIAT的表达沉默降低了 Miiller细胞和RGC的活性、线粒体膜电位以及增殖能力,促进了凋亡;共培养实验表明,Miiller细胞的MIAT表达改变间接地影响视网膜神经元和内皮细胞的功能;机制研究发现,MIAT可通过MIAT/miR-150-5P/VEGF来调控Muller细胞的功能,从而调节眼部神经血管间的相互作用。结论:本研究发现,长链非编码RNA-MIAT是眼部血管病变和神经病变共有的调控因子;MIAT可通过MIAT/miR-150-5P/VEGF信号通路调控视网膜细胞的功能。MIAT有望成为治疗眼部神经血管功能紊乱的新靶点。
[Abstract]:Objective: although blood vessels and nervous systems are functionally different. But they usually have similar functional maintenance mechanism and the same regulatory factors. Neurovascular dysfunction has become the pathogenesis of some vascular diseases and neuropathic diseases. This study aims to elucidate the long chain noncoding RN. A-MIATA. Long non-coding RNA-MIATA). Regulation of neurovascular dysfunction in the eye and its mechanism. Methods: we first detected the process of neurovascular dysfunction by qRT-PCR. Expression of MIAT in animal models of retinopathy and neuropathy as well as retinal vascular and neuronal lesions. The correlation between ocular vascular neuropathy and MIAT expression was clarified. Retinal vascular development, corneal neovascularization (CNV) and diabetic retinopathy (IB4) were used in vivo. Slit lamp microscopy and immunofluorescence staining revealed the regulatory effect of MIAT intervention on ocular neurovascular dysfunction. In vitro, Hoechst staining and PI/Calcein-AM staining were used to reveal the effect of MIAT expression on retinal cell viability and proliferation. The effects of apoptosis and cell interaction; In the aspect of mechanism study, luciferase and qRT-PCR experiments were used to reveal the molecular mechanism of MIAT regulating the function of Muller cells. The expression of MIAT was abnormal in oxygen-induced retinopathy, optic nerve injury and diabetic animal model. The expression of MIAT was abnormal in Miiller cells, endothelial cells, pericytes and neurons under hypoxia or hyperoxia stress. The expression of MIAT was the most obvious in Miiller cells under stress. In vivo, it was found that the silencing of the expression of MIAT inhibited the development of retinal blood vessels in mice. It alleviated corneal neovascularization in alkali burn mice, reduced retina glialization and RGC survival in diabetic mice. The silencing of MIAT expression decreased the activity of Miiller cells and RGC, mitochondrial membrane potential and proliferation ability, and promoted apoptosis. The results of co-culture showed that the expression of MIAT in Miiller cells indirectly affected the function of retinal neurons and endothelial cells. It was found that MIAT/miR-150-5P/VEGF can regulate the function of Muller cells. Conclusion: in this study, long chain noncoding RNA-MIAT is a common regulatory factor for ocular vascular lesions and neuropathy. MIAT can regulate the function of retinal cells through MIAT/miR-150-5P/VEGF signaling pathway. MIAT may be a new target for the treatment of ocular neurovascular dysfunction.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R77

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