顶端极性蛋白复合物在斑马鱼晶状体细胞运动与组织中的功能

发布时间：2018-04-14 23:12

本文选题：斑马鱼 + 转基因　；参考：《浙江大学》2017年博士论文

【摘要】：研究目的:晶状体为眼球屈光间质的重要组成部分,其将光线准确聚焦到视网膜,并通过调节作用看清远近物体,从而形成清晰视力。成熟晶状体是由两类细胞按特定方式排列形成的透明光学结构:前表面的单层立方上皮和内部晶状体纤维细胞,在发育过程中其正确的运动和组织方式是形成这一有序结构的生理基础,任何影响其正确排列的因素都将导致晶状体发育异常,从而导致视力下降甚至致盲。细胞极性为生物体中广泛存在的一种生物学特征,对于胚胎发育、细胞迁移、上皮-间质转化、组织形态维持等诸多生物学事件都起到关键作用。近年来有研究表明,细胞极性在细胞粘连、运动和迁移中具有重要功能,晶状体发育过程中,上皮细胞可发生EMT,表现为细胞极性、细胞间连接消失,上皮细胞转换为间质细胞并具有迁移表型,在此过程中正确的细胞定位、黏附连接和迁移是保证晶状体功能的关键。在细胞极性建立和维持过程中,包括PAR,Crumbs和SCRIB在内的三类高度保守的极性蛋白复合物发挥了重要作用,这些复合物的定位和相互作用对于细胞极性的调节至关重要,但其在晶状体发育过程中的具体作用机制尚未明确。基于以上背景,本研究以斑马鱼为模型,系统地分析了顶端极性蛋白Crb和Par复合物对晶状体发育过程中细胞粘连、运动和组织分化的影响。研究方法:本研究以AB系野生型和顶端极性蛋白Crb2a m289、aPKCλ m567、Pard6gb、fh266突变型斑马鱼为模型,利用体式显微镜照相、冰冻组织切片和免疫组化、高分辨率透射电镜等实验技术研究Crb和Par复合物在斑马鱼晶状体发育过程中的定位、表达时序、两种复合物的动态依赖关系,晶状体细胞状态转化时细胞粘连、运动和组织分化的规律以及极性紊乱对晶状体细胞分化、粘连分子的亚细胞定位重组和细胞运动组织等生物学事件的影响和机制,采用统计学方法定量分析了极性障碍对斑马鱼眼球和晶状体大小、晶状体上皮和纤维细胞数目的影响,并以野生型小鼠为模型,采用免疫组化的方法对不同种属晶状体细胞早期极性蛋白的表达和细胞运动方式进行了探讨。研究结果:研究结果表明,在斑马鱼晶状体发育过程中,Par复合物首先表达,且没有呈现出明显的极性分布,在Crb复合物表达后,才诱导细胞出现极性。当两种复合物丧失功能时,斑马鱼眼球和晶状体发育将出现异常,并影响晶状体上皮和纤维细胞的组织方式,表现为增殖区和分化区的细胞数目出现异常,增殖区细胞提前迁移到晶状体内部分化区,但晶状体细胞的分化不受影响。免疫组化和透射电镜结果表明极性障碍是通过影响细胞粘连的方式导致迁移异常,其可导致细胞粘连的解体从而使细胞运动的能力增加。本研究首次揭示了两种极性蛋白复合物在不同状态晶状体细胞中可调控的复杂动态关系,突破了以往领域内对两种复合物之间呈简单正相关联系的认识,并证实了在不同物种早期晶状体发育过程中,Crb复合物均与细胞绕圆心有序运动迁移的组织方式密切相关的结论。研究结论:本研究在斑马鱼上建立了极性基因突变的模型,首次系统地揭示了顶端极性蛋白在斑马鱼晶状体发育过程中发挥的不同功能以及两种复合物之间的动态可调节性:Par复合物介导细胞粘连的早期形成,Crb复合物介导细胞粘连的显微亚细胞定位,二者协同作用,共同调控晶状体两类细胞的运动和组织方式。本研究为探索晶状体细胞发育的机理建立了良好的实验模型,并为进一步研究晶状体疾病奠定了基础。
[Abstract]:Objective: To study the lens as an important part of the eye refractive medium, the light focusing accurately to the retina, and regulating the see objects near and far to form, clear vision. The lens is composed of two kinds of mature cells in a specific way are arranged to form a transparent optical structure: the anterior surface epithelium and internal lens fiber cells in the course of development, the correct movement and organization is the physiological basis of this form ordered structure, any factors affecting the correct arrangement will cause lens abnormalities, resulting in decreased vision or even blindness. Cell polarity is a kind of biological characteristics widely exist in organisms, for embryonic development, cell migration. Epithelial mesenchymal transition, tissue maintenance and play a key role in many biological events. In recent years studies have shown that cell polarity in cell adhesion. The dynamic migration and has an important function, the process of the development of lens, epithelial cells can occur EMT showed cell polarity, cell-cell junction disappeared, epithelial cells into mesenchymal cells and has the correct positioning of the cell migration phenotype, in this process, adhesion and migration is the key to ensure the lens at the cell pole function. To establish and maintain the process, including PAR, Crumbs and SCRIB, three kinds of highly conserved polarity protein complexes play an important role in localization of these complexes and the interaction is critical for regulation of cell polarity, but in the process of lens development in the specific mechanism is not yet clear. Based on the above background. The zebrafish model, systematic analysis of the apical polarity protein Crb and Par complexes on the development of lens cell adhesion process, effects of exercise and tissue differentiation. Methods: This study Study on AB system of wild type and apical polarity protein Crb2a m289, aPKC Pard6gb, lambda m567, fh266 mutant zebrafish as a model, using the stereo microscope photography, frozen tissue sections and immunohistochemical localization, temporal expression, high resolution transmission electron microscopy and other experimental techniques to study Crb and Par complexes in the process of development in the lens of zebra fish the two kinds of complex dynamic dependence, adhesion of lens cell transformation cell and tissue differentiation, movement patterns and polarity disorder of lens cell differentiation, effects of biological events and subcellular localization of the recombinant adhesion molecule and cell movement organization and mechanism, using statistical methods and quantitative analysis of the size of polar obstacle to zebrafish eye and lens, lens epithelial cells and the effects of fiber number, and wild type mice by immunohistochemical methods in different genera of crystalline Early somatic cell polarity protein expression and cell movement was studied. Results: the results showed that in the process of lens development in zebrafish, Par complex first expression, and no showed polar distribution obviously, the expression of Crb complex, to induce cell polarity. When the two kinds of composite physical loss of function, the zebrafish eye and lens development will appear abnormal, and the influence of lens epithelial and fiber cells of the organization, as the number of cell proliferation and differentiation of the abnormal area, cell proliferative zone early migrating to the interior of the lens differentiation, but differentiation of lens cells was not affected by immunohistochemistry and transmission electron microscopy. The results show that the polar disorder is affected by cell adhesion due to abnormal migration, which can lead to the disintegration of cell adhesion to cell movement. The study shows for the first time The complex dynamic relationship between two kinds of polarity protein complexes in different states in lens cells can be controlled, breaking the previous field in a simple understanding of positive correlation between the two compounds, and confirmed earlier in the process of lens development in different species, Crb complexes were closely related with the cells around the center of the orderly movement the migration organization conclusion. Conclusions: This study established polarity gene mutations in the zebrafish model, for the first time systematically reveals the dynamic between the different function of apical polarity proteins during zebrafish in lens development and two kinds of composites can be adjusted: the early formation of the Par complex mediated cell adhesion, microscopic subcellular localization of Crb complexes mediated cell adhesion, the synergistic effect of the two, two kinds of common regulation of lens cell movement and organization. This study is to explore the crystalline body The mechanism of cell development has established a good experimental model and laid a foundation for further study of lens disease.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R776.1

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