斑馬魚少突膠質(zhì)細(xì)胞調(diào)控因子Lingo1的基因表達(dá)和功能鑒定
發(fā)布時(shí)間：2019-05-27 05:53
【摘要】：復(fù)雜的神經(jīng)系統(tǒng)都是由神經(jīng)元和膠質(zhì)細(xì)胞這兩種主要的細(xì)胞類型構(gòu)成的,而髓鞘化則是一種膠質(zhì)細(xì)胞粘附到神經(jīng)元軸突上并引發(fā)質(zhì)膜極化形成多層髓鞘的包裹過(guò)程。中樞神經(jīng)系統(tǒng)(central nervous system, CNS)由少突膠質(zhì)細(xì)胞負(fù)責(zé)髓鞘化,而外周神經(jīng)系統(tǒng)(peripheral nervous system, PNS)的髓鞘化則是由施旺細(xì)胞完成。常見(jiàn)的中樞神經(jīng)系統(tǒng)脫髓鞘疾病,例如多發(fā)性硬化癥(multiple sclerosis, MS),是一種由中樞神經(jīng)系統(tǒng)遭到免疫系統(tǒng)攻擊而引發(fā)的髓鞘損傷和軸突缺失的神經(jīng)退行性疾病。為了探索中樞神經(jīng)系統(tǒng)脫髓鞘的原因并尋找有效的治療方法,需要進(jìn)一步研究發(fā)育過(guò)程中的髓鞘化和脫髓鞘化。 LINGO-1蛋白是一種由富亮氨酸重復(fù)序列(Leucine-rich repeat, LRR)、免疫球蛋白結(jié)構(gòu)域(immunoglobulin domain, Ig)和軸突生長(zhǎng)抑制蛋白(neurite outgrowth inhibitory protein, Nogo)受體相互作用蛋白1構(gòu)成的中樞神經(jīng)系統(tǒng)跨膜蛋白,是少突膠質(zhì)細(xì)胞分化的負(fù)調(diào)控因子。在多種動(dòng)物模型中,靶向抑制LINGO-1可以促進(jìn)神經(jīng)元存活、軸突再生、少突膠質(zhì)細(xì)胞分化和再髓鞘化。盡管在嚙齒類動(dòng)物模型上的研究加深了對(duì)LINGO-1的了解,但是在斑馬魚(Danio rerio)神經(jīng)發(fā)育和髓鞘化過(guò)程中,其作用還不清楚。本文旨在研究lingo1基因表達(dá)的時(shí)空模式及Lingo1b蛋白在斑馬魚發(fā)育中的作用。 首先我們使用生物信息學(xué)方法分析了斑馬魚lingo1基因的分子結(jié)構(gòu)。相對(duì)哺乳動(dòng)物,斑馬魚同源lingo1有兩個(gè)基因拷貝：lingola和lingo1b。斑馬魚Lingo1b蛋白具有類似哺乳類的蛋白結(jié)構(gòu)并可能在中樞神經(jīng)系統(tǒng)中發(fā)揮作用,在脊椎動(dòng)物中也是高度保守的。利用體外合成lingo1基因的地高辛(digoxin, DIG)標(biāo)記的RNA探針,我們通過(guò)整體原位雜交(whole-mount in situ hybridization, WISH)技術(shù)分析了lingo1基因表達(dá)的時(shí)間和空間模式。通過(guò)實(shí)時(shí)熒光定量PCR (real-time quantitative-polymerase chain reaction, Q-PCR)分析和蛋白免疫印跡(western blot, WB)分析進(jìn)一步發(fā)現(xiàn)斑馬魚lingo1b基因的mRNA表達(dá)始于受精后1天(day post-fertilization, dpf),而蛋白表達(dá)始于2dpf。在斑馬魚lingo1b的反義嗎啡林(morpholino oligonucleotide, MO)基因敲減實(shí)驗(yàn)中,我們首先體外合成了含有MO靶向序列的綠色熒光蛋白(green fluorescent protein, GFP)報(bào)告mRNA序列以及Lingo1b蛋白抗體以分析基因敲減的特異性和效率。對(duì)4dfp斑馬魚lingo1b的Mo1基因敲減后的表型進(jìn)行分析,發(fā)現(xiàn)對(duì)Lingo1b蛋白的表達(dá)抑制會(huì)導(dǎo)致例如黑色素減少、眼睛萎縮和脊椎(spinal cord, SC)彎曲等發(fā)育異常,暗示了其對(duì)神經(jīng)系統(tǒng)發(fā)育的影響。通過(guò)透射電鏡(transmit electron microscopy, TEM)成像和髓鞘的G-ratio分析,我們發(fā)現(xiàn)Mo1敲減后毛特納細(xì)胞軸突(Mauthner axons, MAs)和其它神經(jīng)元軸突的髓鞘變厚并出現(xiàn)了過(guò)早髓鞘化,進(jìn)一分析也暗示lingo1b的敲減能夠促進(jìn)少突膠質(zhì)細(xì)胞的分化與成熟。對(duì)EM圖像的進(jìn)一步分析,我們發(fā)現(xiàn)伴隨著髓鞘的增厚,毛特納細(xì)胞的軸突周長(zhǎng)變小。通過(guò)針對(duì)MAs的整體免疫組化(whole-mount immunohistochemisty, WIHC)標(biāo)記和共聚焦成像分析則進(jìn)一步顯示了lingo1b敲減后MAs的平均直徑下降了,暗示了敲減后引起的過(guò)早髓鞘化可能會(huì)影響初級(jí)運(yùn)動(dòng)神經(jīng)元(primary motor neurons, pMNs)的發(fā)育。我們采用自發(fā)運(yùn)動(dòng)和眼動(dòng)反應(yīng)(optokinetic response, OKR)行為學(xué)分析來(lái)進(jìn)一步研究過(guò)早髓鞘化和運(yùn)動(dòng)神經(jīng)元異常發(fā)育所導(dǎo)致的結(jié)果。圖像分析軟件(Image-Pro Plus, IPP)定量數(shù)據(jù)顯示lingo1b敲減后,單位時(shí)間內(nèi)自發(fā)運(yùn)動(dòng)的累積距離(accumulation distance, Ace Dist)和眼動(dòng)反應(yīng)的頻率均發(fā)生了下降。利用生物信息學(xué)分析,我們從斑馬魚基因組DNA中預(yù)測(cè)并克隆了lingo1b基因的啟動(dòng)子序列,并整合到含有轉(zhuǎn)座酶(TOl2)或巨核酸酶(I-Scel)位點(diǎn)以及熒光報(bào)告序列的表達(dá)載體上。通過(guò)激光共聚焦成像對(duì)顯微注射后的斑馬魚胚胎的熒光信號(hào)模式進(jìn)行了分析,初步構(gòu)建了Lingolb特異性標(biāo)記的轉(zhuǎn)基因斑馬魚品系Tg(lingo1b:EGFP)。 我們的研究結(jié)果顯示,斑馬魚lingo1基因是保守的并在中樞神經(jīng)系統(tǒng)中特異表達(dá)。嗎啡林基因敲減的結(jié)果則顯示斑馬魚Lingo1b蛋白在神經(jīng)系統(tǒng)發(fā)育過(guò)程中,對(duì)于少突膠質(zhì)細(xì)胞分化成熟以及髓鞘化進(jìn)程是一個(gè)重要的負(fù)調(diào)控因子。而形態(tài)學(xué)和行為學(xué)的結(jié)果則提示了由lingo1b基因敲減引起的過(guò)早髓鞘化可能會(huì)影響運(yùn)動(dòng)神經(jīng)元的發(fā)育。這些結(jié)果為深入了解神經(jīng)系統(tǒng)損傷修復(fù)機(jī)制以及以后大規(guī)模篩選潛在的治療脫髓鞘疾病的藥物分子提供了一個(gè)新的平臺(tái)。
[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.
【學(xué)位授予單位】：中國(guó)科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R744.5
本文編號(hào)：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.
【學(xué)位授予單位】：中國(guó)科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R744.5
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 ;Reverse Genetic Approaches in Zebrafish[J];遺傳學(xué)報(bào);2012年09期
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