斑马鱼少突胶质细胞调控因子Lingo1的基因表达和功能鉴定
发布时间：2019-05-27 05:53
【摘要】：复杂的神经系统都是由神经元和胶质细胞这两种主要的细胞类型构成的,而髓鞘化则是一种胶质细胞粘附到神经元轴突上并引发质膜极化形成多层髓鞘的包裹过程。中枢神经系统(central nervous system, CNS)由少突胶质细胞负责髓鞘化,而外周神经系统(peripheral nervous system, PNS)的髓鞘化则是由施旺细胞完成。常见的中枢神经系统脱髓鞘疾病,例如多发性硬化症(multiple sclerosis, MS),是一种由中枢神经系统遭到免疫系统攻击而引发的髓鞘损伤和轴突缺失的神经退行性疾病。为了探索中枢神经系统脱髓鞘的原因并寻找有效的治疗方法,需要进一步研究发育过程中的髓鞘化和脱髓鞘化。 LINGO-1蛋白是一种由富亮氨酸重复序列(Leucine-rich repeat, LRR)、免疫球蛋白结构域(immunoglobulin domain, Ig)和轴突生长抑制蛋白(neurite outgrowth inhibitory protein, Nogo)受体相互作用蛋白1构成的中枢神经系统跨膜蛋白,是少突胶质细胞分化的负调控因子。在多种动物模型中,靶向抑制LINGO-1可以促进神经元存活、轴突再生、少突胶质细胞分化和再髓鞘化。尽管在啮齿类动物模型上的研究加深了对LINGO-1的了解,但是在斑马鱼(Danio rerio)神经发育和髓鞘化过程中,其作用还不清楚。本文旨在研究lingo1基因表达的时空模式及Lingo1b蛋白在斑马鱼发育中的作用。 首先我们使用生物信息学方法分析了斑马鱼lingo1基因的分子结构。相对哺乳动物,斑马鱼同源lingo1有两个基因拷贝：lingola和lingo1b。斑马鱼Lingo1b蛋白具有类似哺乳类的蛋白结构并可能在中枢神经系统中发挥作用,在脊椎动物中也是高度保守的。利用体外合成lingo1基因的地高辛(digoxin, DIG)标记的RNA探针,我们通过整体原位杂交(whole-mount in situ hybridization, WISH)技术分析了lingo1基因表达的时间和空间模式。通过实时荧光定量PCR (real-time quantitative-polymerase chain reaction, Q-PCR)分析和蛋白免疫印迹(western blot, WB)分析进一步发现斑马鱼lingo1b基因的mRNA表达始于受精后1天(day post-fertilization, dpf),而蛋白表达始于2dpf。在斑马鱼lingo1b的反义吗啡林(morpholino oligonucleotide, MO)基因敲减实验中,我们首先体外合成了含有MO靶向序列的绿色荧光蛋白(green fluorescent protein, GFP)报告mRNA序列以及Lingo1b蛋白抗体以分析基因敲减的特异性和效率。对4dfp斑马鱼lingo1b的Mo1基因敲减后的表型进行分析,发现对Lingo1b蛋白的表达抑制会导致例如黑色素减少、眼睛萎缩和脊椎(spinal cord, SC)弯曲等发育异常,暗示了其对神经系统发育的影响。通过透射电镜(transmit electron microscopy, TEM)成像和髓鞘的G-ratio分析,我们发现Mo1敲减后毛特纳细胞轴突(Mauthner axons, MAs)和其它神经元轴突的髓鞘变厚并出现了过早髓鞘化,进一分析也暗示lingo1b的敲减能够促进少突胶质细胞的分化与成熟。对EM图像的进一步分析,我们发现伴随着髓鞘的增厚,毛特纳细胞的轴突周长变小。通过针对MAs的整体免疫组化(whole-mount immunohistochemisty, WIHC)标记和共聚焦成像分析则进一步显示了lingo1b敲减后MAs的平均直径下降了,暗示了敲减后引起的过早髓鞘化可能会影响初级运动神经元(primary motor neurons, pMNs)的发育。我们采用自发运动和眼动反应(optokinetic response, OKR)行为学分析来进一步研究过早髓鞘化和运动神经元异常发育所导致的结果。图像分析软件(Image-Pro Plus, IPP)定量数据显示lingo1b敲减后,单位时间内自发运动的累积距离(accumulation distance, Ace Dist)和眼动反应的频率均发生了下降。利用生物信息学分析,我们从斑马鱼基因组DNA中预测并克隆了lingo1b基因的启动子序列,并整合到含有转座酶(TOl2)或巨核酸酶(I-Scel)位点以及荧光报告序列的表达载体上。通过激光共聚焦成像对显微注射后的斑马鱼胚胎的荧光信号模式进行了分析,初步构建了Lingolb特异性标记的转基因斑马鱼品系Tg(lingo1b:EGFP)。 我们的研究结果显示,斑马鱼lingo1基因是保守的并在中枢神经系统中特异表达。吗啡林基因敲减的结果则显示斑马鱼Lingo1b蛋白在神经系统发育过程中,对于少突胶质细胞分化成熟以及髓鞘化进程是一个重要的负调控因子。而形态学和行为学的结果则提示了由lingo1b基因敲减引起的过早髓鞘化可能会影响运动神经元的发育。这些结果为深入了解神经系统损伤修复机制以及以后大规模筛选潜在的治疗脱髓鞘疾病的药物分子提供了一个新的平台。
[Abstract]:The complex nervous system is composed of two main types of cells of the neuron and the glial cell, and the myeloping is a process of wrapping a colloid cell on the axon of the neuron and inducing the polarization of the plasma membrane to form the multi-layer pulp. The central nervous system (CNS) is made of oligodendrocytes, and the peripheral nervous system (PNS) is made by Schwann cells. The common central nervous system, such as multiple sclerosis, MS, is a neurodegenerative disease that is caused by the attack of the central nervous system by the immune system. In order to explore the causes of the central nervous system defibrination and to find an effective method of treatment, it is necessary to further study the pulpialization and defibrination in the development process. The LINGO-1 protein is a transmembrane egg of the central nervous system consisting of a leucine-rich repeat (LRR), an immunoglobulin domain (Ig) and a neurite outgrowth inhibitory protein (Nogo) receptor interacting protein 1. The white is the negative regulation of the differentiation of oligodendrocytes. in a variety of animal model, targeted inhibition of LINGO-1 may promote neuronal survival, axon regeneration, oligodendrocyte differentiation and re-myeloid differentiation, Although the study on rodent models has deepened the knowledge of the LINGO-1, its role is unclear in the process of the neurodevelopment and the myelopathy of the Zebrafish (Danio ricio). The purpose of this study is to study the spatial and temporal patterns of the expression of the lingo1 gene and the role of the Lingo1b protein in the development of zebrafish First of all, we used the bioinformatics method to analyze the division of the Zebrafish ling1 gene. Substructure. Relative to a mammal, the zebrafish homologous lingo1 has two gene copies: lingola and ling o1b. The zebrafish Lino1b protein has a protein structure similar to that of a mammal and may function in the central nervous system, also in vertebrates The time and space of the expression of the ling1 gene were analyzed by a whole in-situ hybridization (ISH) technique using a digoxin (DIG)-labeled RNA probe for the in vitro synthesis of the ling1 gene. The mRNA expression of the liingo1b gene of the zebrafish was further detected by real-time fluorescence quantitative PCR (Q-PCR) and western blot (WB). Dpf. in the knock-down of the morpheo oligonotide (mo) gene of the zebrafish lingo1b, we first synthesized a green fluorescent protein (gfp) reporter mrna sequence containing the mo-targeting sequence and the lino1b protein antibody to analyze the specificity of the knock-down of the gene. And efficiency. The phenotype of the Mo1 gene of the 4dfp zebrafish lingo1b was analyzed. It was found that the inhibition of the expression of the Lingo1b protein could lead to, for example, the reduction of melanin, the atrophy of the eyes and the abnormal development of the spinal cord (SC), suggesting the development of the nervous system. The effects of Mo1 on the axons (Mathner axons, MAs) and other neuronal axons were found to be thickened and premature by the transmission electron microscopy (TEM) imaging and the G-ratio analysis of the pulp. It is also suggested that the knockdown of lingo1b can promote the differentiation of oligodendrocytes. and maturation. Further analysis of the EM images, we found that with the thickening of the medullary canal, the axons of the hair Turner cells The length was small. The mean diameter of the MAs decreased further by the overall immunohistochemistry (WIHC) labeling and co-focus imaging analysis for MAs, suggesting that the premature myelopathy caused by the knock reduction may affect primary motor netrons, pMNs. We use the behavior analysis of the spontaneous motion and the eye movement response (OKR) to further study the premature and abnormal development of the motoneurons. As a result, the image analysis software (Image-Pro Plus, IPP) quantitative data shows that the cumulative distance (Ace Dist) and the frequency of the eye movement reaction occur in the unit time after the quantitative data of the image analysis software (Image-Pro Plus, IPP) is displayed. With the bioinformatics analysis, the promoter sequence of the lingo1b gene was predicted and cloned from the zebrafish genomic DNA, and the expression of the fluorescent reporter gene (TO2) or the giant nuclease (I-Sel) site and the fluorescent reporter sequence was integrated. The fluorescence signal pattern of the Zebrafish embryos after microinjection was analyzed by laser confocal imaging. FP). Our results show that the zebrafish ling1 gene is conserved and in the central nervous system The results of the knockdown of the morphine-in-lin gene show that the Zebrafish Lino1b protein is an important part of the process of differentiation and maturation of oligodendrocytes and the process of myeloid differentiation in the process of nervous system development. Negative control factors. The results of morphology and behavior suggest that premature mylation caused by the knock-down of the lingo1b gene may affect the exercise god. These results provide an insight into the mechanism of the repair of the nervous system and the potential for large-scale screening of potential drug molecules for the treatment of extramedullary disease.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R744.5
本文编号：2485941
[Abstract]:The complex nervous system is composed of two main types of cells of the neuron and the glial cell, and the myeloping is a process of wrapping a colloid cell on the axon of the neuron and inducing the polarization of the plasma membrane to form the multi-layer pulp. The central nervous system (CNS) is made of oligodendrocytes, and the peripheral nervous system (PNS) is made by Schwann cells. The common central nervous system, such as multiple sclerosis, MS, is a neurodegenerative disease that is caused by the attack of the central nervous system by the immune system. In order to explore the causes of the central nervous system defibrination and to find an effective method of treatment, it is necessary to further study the pulpialization and defibrination in the development process. The LINGO-1 protein is a transmembrane egg of the central nervous system consisting of a leucine-rich repeat (LRR), an immunoglobulin domain (Ig) and a neurite outgrowth inhibitory protein (Nogo) receptor interacting protein 1. The white is the negative regulation of the differentiation of oligodendrocytes. in a variety of animal model, targeted inhibition of LINGO-1 may promote neuronal survival, axon regeneration, oligodendrocyte differentiation and re-myeloid differentiation, Although the study on rodent models has deepened the knowledge of the LINGO-1, its role is unclear in the process of the neurodevelopment and the myelopathy of the Zebrafish (Danio ricio). The purpose of this study is to study the spatial and temporal patterns of the expression of the lingo1 gene and the role of the Lingo1b protein in the development of zebrafish First of all, we used the bioinformatics method to analyze the division of the Zebrafish ling1 gene. Substructure. Relative to a mammal, the zebrafish homologous lingo1 has two gene copies: lingola and ling o1b. The zebrafish Lino1b protein has a protein structure similar to that of a mammal and may function in the central nervous system, also in vertebrates The time and space of the expression of the ling1 gene were analyzed by a whole in-situ hybridization (ISH) technique using a digoxin (DIG)-labeled RNA probe for the in vitro synthesis of the ling1 gene. The mRNA expression of the liingo1b gene of the zebrafish was further detected by real-time fluorescence quantitative PCR (Q-PCR) and western blot (WB). Dpf. in the knock-down of the morpheo oligonotide (mo) gene of the zebrafish lingo1b, we first synthesized a green fluorescent protein (gfp) reporter mrna sequence containing the mo-targeting sequence and the lino1b protein antibody to analyze the specificity of the knock-down of the gene. And efficiency. The phenotype of the Mo1 gene of the 4dfp zebrafish lingo1b was analyzed. It was found that the inhibition of the expression of the Lingo1b protein could lead to, for example, the reduction of melanin, the atrophy of the eyes and the abnormal development of the spinal cord (SC), suggesting the development of the nervous system. The effects of Mo1 on the axons (Mathner axons, MAs) and other neuronal axons were found to be thickened and premature by the transmission electron microscopy (TEM) imaging and the G-ratio analysis of the pulp. It is also suggested that the knockdown of lingo1b can promote the differentiation of oligodendrocytes. and maturation. Further analysis of the EM images, we found that with the thickening of the medullary canal, the axons of the hair Turner cells The length was small. The mean diameter of the MAs decreased further by the overall immunohistochemistry (WIHC) labeling and co-focus imaging analysis for MAs, suggesting that the premature myelopathy caused by the knock reduction may affect primary motor netrons, pMNs. We use the behavior analysis of the spontaneous motion and the eye movement response (OKR) to further study the premature and abnormal development of the motoneurons. As a result, the image analysis software (Image-Pro Plus, IPP) quantitative data shows that the cumulative distance (Ace Dist) and the frequency of the eye movement reaction occur in the unit time after the quantitative data of the image analysis software (Image-Pro Plus, IPP) is displayed. With the bioinformatics analysis, the promoter sequence of the lingo1b gene was predicted and cloned from the zebrafish genomic DNA, and the expression of the fluorescent reporter gene (TO2) or the giant nuclease (I-Sel) site and the fluorescent reporter sequence was integrated. The fluorescence signal pattern of the Zebrafish embryos after microinjection was analyzed by laser confocal imaging. FP). Our results show that the zebrafish ling1 gene is conserved and in the central nervous system The results of the knockdown of the morphine-in-lin gene show that the Zebrafish Lino1b protein is an important part of the process of differentiation and maturation of oligodendrocytes and the process of myeloid differentiation in the process of nervous system development. Negative control factors. The results of morphology and behavior suggest that premature mylation caused by the knock-down of the lingo1b gene may affect the exercise god. These results provide an insight into the mechanism of the repair of the nervous system and the potential for large-scale screening of potential drug molecules for the treatment of extramedullary disease.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R744.5
【参考文献】
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1 ;Reverse Genetic Approaches in Zebrafish[J];遗传学报;2012年09期
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