Lox11基因敲除小鼠对慢性高眼压损伤的易感性研究
发布时间：2019-07-02 09:29
【摘要】：青光眼是世界第二大致盲性疾病,临床表现为进行性视野丢失及视乳头凹陷。高眼压是青光眼视神经病变最主要的病因,机械损伤可最终造成RGC凋亡、视神经萎缩,即青光眼视神经病变。视乳头是眼球结构的一个“薄弱点”,由眼内压产生的机械力作用于视乳头周围的眼球底部,并在致密的巩膜和相对薄弱的视乳头的移行处被放大,故视乳头处的视神经对眼内压的变化更为敏感,是青光眼视神经病变首先累及的位点。视神经筛板及球后环视乳头组织内的弹性纤维赋予了组织良好的顺应性和可塑性,可一定程度地缓冲眼内压波动对视神经产生的影响。弹性蛋白是构成弹性纤维的主要成分,LOXL1是催化弹性蛋白聚合再生的关键酶,对维持弹性纤维稳态至关重要。假性色素剥脱青光眼(PEXG)患者的眼压难控制并且对青光眼视神经病变较敏感,在患者的筛板组织内发现了广泛的弹性纤维降解现象。近几年的研究发现：LOXL1的基因表达缺陷与高眼压同为PEXG的重要危险因素。那么,这两个因素对青光眼视神经病变的进展是否具有协同作用呢?弹性蛋白是否是这二者共同的作用点呢?作用的机制又是怎样的呢?为了探讨以上问题,我们采用了Loxl1基因敲除小鼠(简称Loxl1-/-)作为研究载体,WT小鼠作为对照,利用前房注射微粒珠技术给予慢性高眼压损伤,比较小鼠对损伤的易感性,观察弹性蛋白在病变中的表现。 本研究是中美联合培养博士的合作项目,得到了国家留学基金委和美国国立卫生院的大力支持。研究的贡献是：巩固了微粒珠手术建立小鼠慢性高眼压这一新技术的应用；丰富了对Loxl1-/-小鼠眼部表型的研究；为PEXG的病因机制提供了一个新的研究思路。 本论文由四部分组成。第一章前言首先提到弹性纤维对维系组织顺应性和可塑性的至关重要,概述了弹性纤维的合成降解过程以及相关的病理制,强调弹性纤维稳态的维持需要LOXL1的参与。而后提出弹性纤维参与了青光眼视神经病变的生物力学机制,LOXL1缺陷使弹性纤维从稳态转为紊态,与高眼压一同成为PEXG的主要病因。最后对Loxl1-/-小鼠以及小鼠高眼压模型的发展概况进行了简要介绍。而后,提出本文的研究方向和研究特色。 第二章所述工作为选择前房注射微粒珠的手术技术建立小鼠慢性高眼压模型。本实验各选用25只C57/BL6(野生型和)Loxl1-/-小鼠,前房注射2μl微粒珠(6μm)+3μl粘弹剂,以tonolab小鼠专用眼压测量机监测小鼠术后眼压,并且观察眼部体征,观察时间为术后56天。微粒珠手术成功建立了小鼠慢性高眼压模型,两组实验动物术后眼压变化的总体特征是相近的,WT眼压的波动相对较大。 第三章中,我们在56天观察期满后取材,测量术眼眼轴变化,评估术眼视乳头凹陷程度,并且对RGC轴突进行计数,观察了视神经内的病理形态,明确微粒珠手术所造成的慢性高眼压在小鼠眼内导致了类青光眼视神经病变。并且我们发现：Lox11-/-小鼠的眼球的扩张程度更明显,眼轴的增长呈现高度眼压依赖性,视乳头凹陷、RGC轴突的病变程度都比WT严重,可见Lox11-/-小鼠对慢性高眼压损伤更敏感。 第四章工作利用免疫荧光染色和蛋白印迹学技术对弹性蛋白在慢性高眼压下的表现进行了研究。我们发现：Lox11-/-小鼠眼球后极部及球后环视乳头组织内的弹性纤维结构排列疏松,在慢性高眼压损伤下存在明显的弹性蛋白再生障碍及广泛降解现象,同时眼内单体弹性蛋白及弹性蛋白降解产物堆积,印证了形态学的改变。 研究认为：Lox11-/-小鼠眼内LOXL1表达缺陷使其在慢性高眼压损伤下合并出现弹性蛋白的再生障碍以及异常降解,眼球壁以及环视乳头组织的顺应性和可塑性下降,使得Lox11-/-小鼠的视神经更易受到机械性损伤,弹性蛋白降解产物累积并发挥生物活性,可能进一步加剧视神经病变程度。
[Abstract]:Glaucoma is the second-largest blindness disease in the world. The clinical manifestation is progressive visual field loss and papilla depression. High intraocular pressure (IOP) is the main cause of the optic neuropathy of the glaucoma, and the mechanical injury can ultimately cause the apoptosis of RGC and the atrophy of the optic nerve, that is, the optic neuropathy of the glaucoma. if the nipple is a "weak point" of the eye structure, the mechanical force generated by the internal pressure of the eye acts on the bottom of the eye around the nipple and is enlarged at the transitional portion of the dense sclera and the relatively weak visual nipple, so that the optic nerve at the nipple is more sensitive to the change in the internal pressure of the eye, It is the first involved site of the optic neuropathy of the glaucoma. The elastic fibers in the optic nerve screen plate and the post-spherical ring-optic papilla tissue give the tissue good compliance and plasticity, and the effect of the internal pressure fluctuation on the optic nerve can be buffered to a certain extent. The elastic protein is the main component of the elastic fiber, and the LOXL1 is the key enzyme for catalyzing the polymerization and regeneration of the elastic protein, and is essential for maintaining the steady state of the elastic fiber. It is difficult to control the intraocular pressure in the patients with pseudopigmentation glaucoma (PEXG) and is more sensitive to the optic neuropathy of the glaucoma, and a wide range of elastic fiber degradation is found in the patient's sieve plate tissue. In recent years, it has been found that LOXL1 gene expression defect and high intraocular pressure are an important risk factor for PEXG. So, does the two factors have a synergistic effect on the progression of the glaucoma optic neuropathy? Is the elastin a common point of action for both? What is the mechanism of action? In order to study the above problems, we used the Loxl1 knockout mouse (Loxl1-/-) as the research carrier and WT mice as the control, and using the anterior chamber injection of the particle bead technique to give the chronic high intraocular pressure injury, and compared the susceptibility of the mouse to the injury, The expression of elastin in the lesion was observed. The research is a cooperation project of the joint culture of China and the United States, and is supported by the NSFC and the National Health Center of the United States. The contribution of the study is to consolidate the application of the new technique for establishing the chronic high intraocular pressure in mice by the microbead operation, and to enrich the research on the eye phenotype of the Loxl1-/-mouse, and to provide a new research for the etiological mechanism of the PEXG. Road. This paper is composed of four parts The first part of the first chapter is to mention the importance of the elastic fiber in the maintenance of the compliance and plasticity of the tissue. The process of the synthesis and degradation of the elastic fiber and the related pathological system are introduced, and the maintenance of the steady state of the elastic fiber is emphasized. The LOXL1 is emphasized. The results show that the elastic fiber is involved in the biomechanical mechanism of the optic neuropathy of the glaucoma. The LOXL1 defect changes the elastic fiber from the steady state to the low-pressure state, and becomes the main body of the PEXG with the high intraocular pressure. The development of the high intraocular pressure model of Loxl1-/-mouse and mouse was also reviewed. In this paper, the research direction and the research of this paper are put forward. To study the features of the study. In the second chapter, the operative technique for the selection of the anterior chamber injection of the microbeads was used to establish the mice's chronic In this experiment,25 C57/ BL6 (wild type and) Loxl1-/-mice were selected, and the anterior chamber was injected with 2. m The model of chronic high intraocular pressure in mice was successfully established after operation for 56 days. The overall characteristics of the changes of intraocular pressure in two groups of experimental animals were similar, and the wave of intraocular pressure in the two groups was similar. In the third chapter, we observed the changes of the eye-eye axis after the expiration of the 56-day observation, and evaluated the degree of the eye-to-eye degree of the eye, and the RGC axons were counted, and the visual and visual effects were observed. The internal pathological form, the chronic high intraocular pressure caused by the fine particle-bead operation resulted in the blue-green in the mouse's eye. Optic neuropathy was found. And we found that the degree of dilation of the eye of the Lox11-/-mouse is more pronounced, the growth of the eye is highly dependent on the ocular pressure, the degree of lesion of the RGC axon is more severe than that of the WT, and the Lox11-/-mice are chronic high. Chapter 4: Immunofluorescence and Western Blot in the Treatment of Chronic High-intraocular Pressure The results of this study were studied. We found that the elastic fiber structure in the posterior polar part of the eye and the post-spherical ring of the eye of the mouse was loose, and there were obvious elastic protein regeneration and extensive degradation under the condition of chronic high intraocular pressure, and the elastic protein and the elastic protein of the intraocular monomer. the degradation products of the sex protein are stacked, The changes of the morphology were confirmed. The study found that the expression of LOXL1 in the Lox11-/-mice caused the combination of the regeneration barrier and the abnormal degradation of the elastin under the injury of chronic high intraocular pressure, the eye wall and the group of the ring-optic papilla. The compliance and plasticity of the woven fabric are reduced, so that the optic nerve of the Lox11-/-mouse is more susceptible to mechanical damage, the degradation products of the elastin are accumulated and the biological activity is exerted,
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R775
本文编号：2508807
[Abstract]:Glaucoma is the second-largest blindness disease in the world. The clinical manifestation is progressive visual field loss and papilla depression. High intraocular pressure (IOP) is the main cause of the optic neuropathy of the glaucoma, and the mechanical injury can ultimately cause the apoptosis of RGC and the atrophy of the optic nerve, that is, the optic neuropathy of the glaucoma. if the nipple is a "weak point" of the eye structure, the mechanical force generated by the internal pressure of the eye acts on the bottom of the eye around the nipple and is enlarged at the transitional portion of the dense sclera and the relatively weak visual nipple, so that the optic nerve at the nipple is more sensitive to the change in the internal pressure of the eye, It is the first involved site of the optic neuropathy of the glaucoma. The elastic fibers in the optic nerve screen plate and the post-spherical ring-optic papilla tissue give the tissue good compliance and plasticity, and the effect of the internal pressure fluctuation on the optic nerve can be buffered to a certain extent. The elastic protein is the main component of the elastic fiber, and the LOXL1 is the key enzyme for catalyzing the polymerization and regeneration of the elastic protein, and is essential for maintaining the steady state of the elastic fiber. It is difficult to control the intraocular pressure in the patients with pseudopigmentation glaucoma (PEXG) and is more sensitive to the optic neuropathy of the glaucoma, and a wide range of elastic fiber degradation is found in the patient's sieve plate tissue. In recent years, it has been found that LOXL1 gene expression defect and high intraocular pressure are an important risk factor for PEXG. So, does the two factors have a synergistic effect on the progression of the glaucoma optic neuropathy? Is the elastin a common point of action for both? What is the mechanism of action? In order to study the above problems, we used the Loxl1 knockout mouse (Loxl1-/-) as the research carrier and WT mice as the control, and using the anterior chamber injection of the particle bead technique to give the chronic high intraocular pressure injury, and compared the susceptibility of the mouse to the injury, The expression of elastin in the lesion was observed. The research is a cooperation project of the joint culture of China and the United States, and is supported by the NSFC and the National Health Center of the United States. The contribution of the study is to consolidate the application of the new technique for establishing the chronic high intraocular pressure in mice by the microbead operation, and to enrich the research on the eye phenotype of the Loxl1-/-mouse, and to provide a new research for the etiological mechanism of the PEXG. Road. This paper is composed of four parts The first part of the first chapter is to mention the importance of the elastic fiber in the maintenance of the compliance and plasticity of the tissue. The process of the synthesis and degradation of the elastic fiber and the related pathological system are introduced, and the maintenance of the steady state of the elastic fiber is emphasized. The LOXL1 is emphasized. The results show that the elastic fiber is involved in the biomechanical mechanism of the optic neuropathy of the glaucoma. The LOXL1 defect changes the elastic fiber from the steady state to the low-pressure state, and becomes the main body of the PEXG with the high intraocular pressure. The development of the high intraocular pressure model of Loxl1-/-mouse and mouse was also reviewed. In this paper, the research direction and the research of this paper are put forward. To study the features of the study. In the second chapter, the operative technique for the selection of the anterior chamber injection of the microbeads was used to establish the mice's chronic In this experiment,25 C57/ BL6 (wild type and) Loxl1-/-mice were selected, and the anterior chamber was injected with 2. m The model of chronic high intraocular pressure in mice was successfully established after operation for 56 days. The overall characteristics of the changes of intraocular pressure in two groups of experimental animals were similar, and the wave of intraocular pressure in the two groups was similar. In the third chapter, we observed the changes of the eye-eye axis after the expiration of the 56-day observation, and evaluated the degree of the eye-to-eye degree of the eye, and the RGC axons were counted, and the visual and visual effects were observed. The internal pathological form, the chronic high intraocular pressure caused by the fine particle-bead operation resulted in the blue-green in the mouse's eye. Optic neuropathy was found. And we found that the degree of dilation of the eye of the Lox11-/-mouse is more pronounced, the growth of the eye is highly dependent on the ocular pressure, the degree of lesion of the RGC axon is more severe than that of the WT, and the Lox11-/-mice are chronic high. Chapter 4: Immunofluorescence and Western Blot in the Treatment of Chronic High-intraocular Pressure The results of this study were studied. We found that the elastic fiber structure in the posterior polar part of the eye and the post-spherical ring of the eye of the mouse was loose, and there were obvious elastic protein regeneration and extensive degradation under the condition of chronic high intraocular pressure, and the elastic protein and the elastic protein of the intraocular monomer. the degradation products of the sex protein are stacked, The changes of the morphology were confirmed. The study found that the expression of LOXL1 in the Lox11-/-mice caused the combination of the regeneration barrier and the abnormal degradation of the elastin under the injury of chronic high intraocular pressure, the eye wall and the group of the ring-optic papilla. The compliance and plasticity of the woven fabric are reduced, so that the optic nerve of the Lox11-/-mouse is more susceptible to mechanical damage, the degradation products of the elastin are accumulated and the biological activity is exerted,
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R775
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