斑马鱼视网膜发育及Crb2b对光感受器细胞影响的研究

发布时间：2018-05-29 05:45

本文选题：斑马鱼 + 视网膜　；参考：《吉林大学》2013年博士论文

【摘要】：视网膜色素变性是一种遗传性致盲眼病，目前全世界范围内发病率为19-27/100，000，视网膜光感受器细胞进行性死亡是导致视力丧失的主要原因。光感受器细胞是终末分化的神经元，它们不能再生，一旦丧失就无法替代。因而关于视网膜光感受器细胞退变机制的研究非常重要。自1981年美国俄勒冈大学著名遗传学家George streisinger关于斑马鱼人工雌核发育的研究在《Nature》发表后，斑马鱼作为研究遗传发育的实验动物模型开始引起科学家的极大关注。目前斑马鱼已成为最受重视的脊椎动物发育生物学模型之一。斑马鱼的视网膜感光细胞以视锥细胞为主，在一些视网膜疾病的研究上具有独特优势，比如视网膜色素变性、年龄相关性黄斑变性等，选用斑马鱼作为模式动物极佳。 Crumbs蛋白复合物是上皮极性的一个关键调节物。Crumbs蛋白复合物高度保守，这个复合物中主要的成员-Crumbs(Crb)最初在黑腹果蝇中被发现，对果蝇正常的光感受器细胞形态形成及粘着小带的形成和维持十分重要。在果蝇中只有一种Crb，在脊椎动物中有三种CRB-CRB1、CRB2和CRB3。 CRB1基因突变与人类多种常染色体隐性遗传性视网膜变性相关，包括视网膜色素变性和Leber氏先天性黑蒙。目前对CRB1突变体尚未发现明确的基因型-表型之间的关联，说明Crumbs蛋白复合物的其它成分可能也影响疾病的严重程度。斑马鱼有5种Crb，它们分别是Crb1、Crb2a、Crb2b、Crb3a和Crb3b。在斑马鱼中，两个Crb2基因(Crb2a和Crb2b)与视网膜的发育和形态学缺陷相关。Crb2b主要在斑马鱼视网膜视锥细胞内节表达，Crb2b的细胞外区域介导细胞间粘附。Crb2b在维持光感受器细胞层完整性及稳定视锥细胞马赛克结构平面构建上发挥重要的作用。斑马鱼Crb2b功能与人类CRB1功能相似，研究Crb2b对斑马鱼光感受器细胞的影响有助于进一步揭示视网膜色素变性等退变性视网膜病变的发病机制。精确地计数视网膜细胞在时间和空间上的分布是研究视网膜发育及退变的基础。每个视网膜细胞只有一个细胞核，因此最直接计数视网膜细胞的方法是在光学显微镜下计数细胞核的数目。本论文首先结合JB-4塑胶树脂包埋和福尔根染色的方法观察斑马鱼视网膜细胞核。通过分析JB-4切片厚度、盐酸水解程度及Schiff反应条件对细胞核染色的影响，寻找一个最优地能够敏感且特异染色斑马鱼视网膜细胞核的方法。实验结果显示：盐酸浓度越高、浸润时间越长，染色程度越强；盐酸浓度越低、浸润时间越短，染色程度越弱。低浓度的盐酸可以通过延长浸润时间而提高染色强度。染色强度随着Schiff浸润时间的延长而增加，2小时达到最强染色。在2μm组织切片中单个细胞核比在3μm组织切片、4μm组织切片及6μm组织切片中显示出更清晰的核边界。在2μm厚经福尔根染色的JB-4切片中，视锥细胞中的常染色质区域能够清晰地和异染色质区域区分开。与亚甲天蓝II和核固红染色相比，福尔根染色特异性和稳定性更强。福尔根染色可以和标本包埋原位杂交技术相兼容。通过实验结果分析发现了一个最优地能够敏感且特异染色斑马鱼视网膜细胞核的方法。这个简单可靠的染色方法可以用于细胞计数及检测视锥细胞染色质整体结构，与标本包埋原位杂交技术相结合可同时观察细胞核和mRNA表达。本论文其次通过计数成年野生型斑马鱼视网膜神经节细胞、内核层细胞、水平细胞、视锥细胞及视杆细胞在视网膜不同区域内的细胞数目，通过比较不同年龄斑马鱼之间相同视网膜区域细胞数目的变化及某一特定年龄斑马鱼不同视网膜区域之间细胞数目的变化探讨野生型斑马鱼视网膜发育的特点。实验结果显示：野生型斑马鱼视网膜神经节细胞、内核层细胞、水平细胞、视杆细胞及视锥细胞在视网膜各个区域排列规整。斑马鱼早期视网膜各类细胞发育从周边部开始，逐渐向中央及两侧延伸，最终上形成特殊的空间分布特点：除视杆细胞外各类细胞视网膜中心区细胞数目最多，向两侧逐渐减少形成正态分布；视杆细胞视网膜前部-后部方向后部旁中心区细胞数目最多，向两侧逐渐减少；腹部-背部方向背部旁中心区细胞数目最多，向两侧逐渐减少形成偏正态分布。野生型斑马鱼视网膜细胞在时间上有一定的分布规律：伴随着斑马鱼的年龄增长，视网膜细胞总体呈现下降趋势，视杆细胞在斑马鱼生长早期局部区域呈现上升趋势。该实验结果为进一步揭示视网膜发育机制及研究退变性视网膜病变的发病机制提供必要的基础数据。另外，本实验结果显示斑马鱼随年龄增长光感受器细胞整体呈现下降趋势，晚期下降最为明显。人类年龄相关性黄斑变性是随年龄增长而进展的的光感受器细胞退变性疾病。通过本研究视网膜视锥细胞随年龄增长逐渐死亡，特别是到晚期视锥细胞大量死亡提供的基础数据可进一步揭示年龄相关性性黄斑变性的发病机制。本论文最后计数两个不同年龄阶段pt108b成年斑马鱼视网膜不同区域细胞的数目，观察pt108b斑马鱼视网膜细胞的空间分布；并通过与同年龄野生型斑马鱼相同视网膜区域细胞数目及视网膜细胞空间分布比较探讨Crb2b对视网膜光感受器细胞的影响。实验结果显示：pt108b转基因斑马鱼视网膜各类细胞在空间上并无像野生型斑马鱼一样规律的分布。pt108b转基因斑马鱼视网膜各类细胞分层破坏，细胞间排列松散，视锥细胞排列极其稀疏；特别是生长晚期细胞排列紊乱，个别区域视锥细胞完全丧失。pt108b转基因斑马鱼不同时期视网膜各个区域视锥细胞数目较同龄野生型斑马鱼均显著下降；且视网膜视锥细胞数目随年龄增长显著下降，至晚期视锥细胞数目急剧减少，个别区域视锥细胞几乎完全消失。但生长晚期除视锥细胞外其它细胞数目在视网膜局部区域呈现不同程度的增多，，特别是视杆细胞在视网膜绝大多数区域均显著增多。实验结果说明Crb2b是维持成年斑马鱼视网膜各类细胞正常空间分布及视锥细胞存活的关键蛋白。Crb2b功能抑制可以导致成年斑马鱼视网膜视锥细胞死亡特别是生长晚期视锥细胞大量死亡以及神经节细胞、内核层细胞、水平细胞及视杆细胞在晚期视网膜局部区域不同程度的增生。pt108b转基因斑马鱼由于Crb2b功能抑制可以导致视网膜视锥细胞进行性死亡，逐渐进展的光感受器细胞死亡是遗传性视网膜色素变性显著的特征。该动物模型有助于进一步深入研究视网膜色素变性的发病机制。
[Abstract]:Retinitis pigmentosa is a hereditary blinding ophthalmopathy. The incidence of the disease is 19-27/100000 worldwide. The progressive death of the retina photoreceptor cells is the main cause of visual loss. The photoreceptor cells are terminally differentiated neurons. They can not be regenerated and can not be replaced once they are lost. Therefore, the retinal light is not replaceable. The research on the mechanism of receptor cell degeneration is very important.
Since 1981, George Streisinger, a famous geneticist at the University of Oregon, published a study on zebrafish artificial ethe development of zebrafish, the zebrafish as an experimental animal model for genetic development began to arouse great attention of scientists. At present zebrafish has become one of the most important biological models for vertebrate development. The retina photosensitive cells of zebrafish are dominated by cone cells and have unique advantages in the study of some retinal diseases, such as retinitis pigmentosa, age related macular degeneration, and the selection of zebrafish as a model animal.
The Crumbs protein complex is a key regulator of the epithelial polarity, a key regulator of the.Crumbs protein complex, which is highly conserved. The main member of the complex, -Crumbs (Crb), was first found in the Drosophila melanogaster, which is important for the formation and maintenance of the normal photoreceptor cell morphology and the formation and maintenance of the sticky band. In the Drosophila, there is only one Crb in the fruit fly. There are three kinds of CRB-CRB1, CRB2 and CRB3. in vertebrates
The CRB1 gene mutation is associated with a variety of autosomal recessive retinal degeneration, including retinitis pigmentosa and Leber's congenital melanoma. There is no clear genotype phenotype association for CRB1 mutants, indicating that other components of the Crumbs protein complex may also affect the severity of the disease.
Zebrafish has 5 kinds of Crb, which are Crb1, Crb2a, Crb2b, Crb3a and Crb3b. in zebrafish. The two Crb2 genes (Crb2a and Crb2b) are associated with the retinal development and morphological defects..Crb2b is mainly expressed in the retina of the retina of the zebrafish retina. The extracellular domain mediates the intercellular adhesion in the maintenance of the photoreceptor cell layer. The Crb2b function of zebrafish is similar to that of human CRB1. The study of the effect of Crb2b on the photoreceptor cells of zebrafish can help to further reveal the pathogenesis of retinitis pigmentosa, such as retinitis pigmentosa.
Accurately counting the distribution of retinal cells in time and space is the basis for studying retinal development and degeneration. Each retinal cell has only one nucleus, so the most direct method of counting the retinal cells is to count the number of nuclei under an optical microscope. This paper first combined with JB-4 plastic resin embedding and formaldehyde staining. The effects of JB-4 slice thickness, hydrochloric acid hydrolysis degree and Schiff reaction condition on the cell nucleus staining were analyzed. The optimal method for sensitive and specific staining of zebrafish retinal nuclei was found. The results showed that the higher the concentration of salt acid, the longer the infiltration time, the degree of dyeing. The stronger the concentration, the lower the concentration of hydrochloric acid, the shorter the infiltration time, the weaker the dyeing degree. The low concentration of hydrochloric acid can increase the dyeing strength by prolonging the time of infiltration. The intensity of dyeing increases with the prolongation of the time of Schiff infiltration, and the strongest dyeing is reached at 2 hours. The single cell nucleus of the tissue section of 2 mu is compared to 3 mu m, 4 mu m tissue section and 6 mu. The M tissue section shows a clearer nuclear boundary. In the JB-4 section stained by the 2 m thick, the normal chromatin region in the cone cells can be clearly separated from the heterochromatin region. An optimal method for sensitive and specific staining of zebrafish retinal nuclei is found through experimental results. This simple and reliable staining method can be used for cell counting and detection of the overall structure of the cone cell chromatin, combined with the embedded in situ hybridization technique. The expression of nucleus and mRNA.
This thesis is followed by counting the number of cells in the retinal ganglion cells, core layer cells, horizontal cells, cone cells and rod cells in different retina of the retina of adult wild zebrafish, by comparing the changes in the number of cells in the same retina region between different age zebrafish and the different viewing nets of a certain age of a certain age zebrafish. The retinal ganglion cells of wild zebrafish, core layer cells, horizontal cells, rod cells and cone cells arranged regularly in various regions of the retina, and the development of various cells in the early retina of zebrafish from the periphery of the wild zebrafish. At the beginning, it gradually extends to the central and both sides, and finally forms a special spatial distribution feature: the number of cells in the central area of retina of various cells except the rod cells is the most, and the normal distribution is gradually reduced to both sides; the number of cells in the center of the posterior part of the retina of the rod cells is the most, and the abdomen decreases gradually to both sides. In the back, the number of cells in the central region of the back is the most, and the distribution of the retinal cells in the wild type zebrafish has a certain distribution rule. With the age of zebrafish growth, the retinal cells generally decline, and the rod cell is up in the early local area of the zebrafish. The experimental results provide necessary basic data to further reveal the mechanism of retinal development and the pathogenesis of degenerative retinopathy. In addition, the results of this experiment show that the overall decline of the photoreceptor cells in zebrafish with age increases, and the late decline is most obvious. The progressive death of retinal cone cells with age, especially the basic data provided by the massive death of advanced cone cells in this study, can further reveal the pathogenesis of age-related macular degeneration.
At the end of this paper, the number of cells in different regions of pt108b adult zebrafish pt108b was counted and the spatial distribution of pt108b zebrafish retina cells was observed. The photoreceptor of the retina was compared with the number of cells in the same retina region and the spatial distribution of retinal cells in the same age of the wild zebrafish of the same age. The experimental results showed that the pt108b transgenic zebrafish retina had no spatial distribution of various cells in the retina of the retina like wild zebrafish. The cells in the retina of the.Pt108b transgenic zebrafish were delaminated, the cells were loosely arranged and the cones were very sparse, especially in the late growth of the cells. The number of cone cells in each region of the retina of.Pt108b transgenic zebrafish was significantly lower than that of the same age wild zebrafish, and the number of retinal cone cells decreased significantly with age, and the number of late cone cells decreased dramatically, and the cone cells in a few regions almost completely disappeared. But the number of other cells outside the cone cells in the late growth stage increased in different degrees in the retina, especially in the vast majority of the retina. The results showed that Crb2b was the key protein.Crb2b to maintain the normal spatial distribution of various cells in adult zebrafish retina and the survival of the cones. Functional inhibition can lead to the death of retinal cone cells in adult zebrafish, especially in late growing cones, as well as ganglion cells, core layer cells, horizontal cells, and rod cells in different degrees in the local area of the late retina,.Pt108b transgenic zebra fish can lead to retina due to the inhibition of Crb2b function. The progressive death of the photoreceptor cells is a significant feature of hereditary retinitis pigmentosa. This animal model is helpful to further study the pathogenesis of retinitis pigmentosa.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R774.1

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