本文選題：斑馬魚 + 轉(zhuǎn)基因��； 參考：《浙江大學》2017年博士論文

【摘要】：研究目的:晶狀體為眼球屈光間質(zhì)的重要組成部分,其將光線準確聚焦到視網(wǎng)膜,并通過調(diào)節(jié)作用看清遠近物體,從而形成清晰視力。成熟晶狀體是由兩類細胞按特定方式排列形成的透明光學結(jié)構(gòu):前表面的單層立方上皮和內(nèi)部晶狀體纖維細胞,在發(fā)育過程中其正確的運動和組織方式是形成這一有序結(jié)構(gòu)的生理基礎(chǔ),任何影響其正確排列的因素都將導致晶狀體發(fā)育異常,從而導致視力下降甚至致盲。細胞極性為生物體中廣泛存在的一種生物學特征,對于胚胎發(fā)育、細胞遷移、上皮-間質(zhì)轉(zhuǎn)化、組織形態(tài)維持等諸多生物學事件都起到關(guān)鍵作用。近年來有研究表明,細胞極性在細胞粘連、運動和遷移中具有重要功能,晶狀體發(fā)育過程中,上皮細胞可發(fā)生EMT,表現(xiàn)為細胞極性、細胞間連接消失,上皮細胞轉(zhuǎn)換為間質(zhì)細胞并具有遷移表型,在此過程中正確的細胞定位、黏附連接和遷移是保證晶狀體功能的關(guān)鍵。在細胞極性建立和維持過程中,包括PAR,Crumbs和SCRIB在內(nèi)的三類高度保守的極性蛋白復(fù)合物發(fā)揮了重要作用,這些復(fù)合物的定位和相互作用對于細胞極性的調(diào)節(jié)至關(guān)重要,但其在晶狀體發(fā)育過程中的具體作用機制尚未明確。基于以上背景,本研究以斑馬魚為模型,系統(tǒng)地分析了頂端極性蛋白Crb和Par復(fù)合物對晶狀體發(fā)育過程中細胞粘連、運動和組織分化的影響。研究方法:本研究以AB系野生型和頂端極性蛋白Crb2a m289、aPKCλ m567、Pard6gb、fh266突變型斑馬魚為模型,利用體式顯微鏡照相、冰凍組織切片和免疫組化、高分辨率透射電鏡等實驗技術(shù)研究Crb和Par復(fù)合物在斑馬魚晶狀體發(fā)育過程中的定位、表達時序、兩種復(fù)合物的動態(tài)依賴關(guān)系,晶狀體細胞狀態(tài)轉(zhuǎn)化時細胞粘連、運動和組織分化的規(guī)律以及極性紊亂對晶狀體細胞分化、粘連分子的亞細胞定位重組和細胞運動組織等生物學事件的影響和機制,采用統(tǒng)計學方法定量分析了極性障礙對斑馬魚眼球和晶狀體大小、晶狀體上皮和纖維細胞數(shù)目的影響,并以野生型小鼠為模型,采用免疫組化的方法對不同種屬晶狀體細胞早期極性蛋白的表達和細胞運動方式進行了探討。研究結(jié)果:研究結(jié)果表明,在斑馬魚晶狀體發(fā)育過程中,Par復(fù)合物首先表達,且沒有呈現(xiàn)出明顯的極性分布,在Crb復(fù)合物表達后,才誘導細胞出現(xiàn)極性。當兩種復(fù)合物喪失功能時,斑馬魚眼球和晶狀體發(fā)育將出現(xiàn)異常,并影響晶狀體上皮和纖維細胞的組織方式,表現(xiàn)為增殖區(qū)和分化區(qū)的細胞數(shù)目出現(xiàn)異常,增殖區(qū)細胞提前遷移到晶狀體內(nèi)部分化區(qū),但晶狀體細胞的分化不受影響。免疫組化和透射電鏡結(jié)果表明極性障礙是通過影響細胞粘連的方式導致遷移異常,其可導致細胞粘連的解體從而使細胞運動的能力增加。本研究首次揭示了兩種極性蛋白復(fù)合物在不同狀態(tài)晶狀體細胞中可調(diào)控的復(fù)雜動態(tài)關(guān)系,突破了以往領(lǐng)域內(nèi)對兩種復(fù)合物之間呈簡單正相關(guān)聯(lián)系的認識,并證實了在不同物種早期晶狀體發(fā)育過程中,Crb復(fù)合物均與細胞繞圓心有序運動遷移的組織方式密切相關(guān)的結(jié)論。研究結(jié)論:本研究在斑馬魚上建立了極性基因突變的模型,首次系統(tǒng)地揭示了頂端極性蛋白在斑馬魚晶狀體發(fā)育過程中發(fā)揮的不同功能以及兩種復(fù)合物之間的動態(tài)可調(diào)節(jié)性:Par復(fù)合物介導細胞粘連的早期形成,Crb復(fù)合物介導細胞粘連的顯微亞細胞定位,二者協(xié)同作用,共同調(diào)控晶狀體兩類細胞的運動和組織方式。本研究為探索晶狀體細胞發(fā)育的機理建立了良好的實驗?zāi)Ｐ?并為進一步研究晶狀體疾病奠定了基礎(chǔ)。
[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.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R776.1

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