荧光超分辨显微成像用于研究卵母细胞减数分裂过程

发布时间：2018-01-25 03:38

本文关键词： 随机光重建显微卵母细胞动力蛋白微管蛋白染色体　出处：《深圳大学》2017年硕士论文　论文类型：学位论文

【摘要】：非整倍性(aneuploidy)是人类染色体异常中最常见的类型,生殖细胞非整倍性是引起人类智力低下、先天畸形和妊娠失败的主要原因,体细胞非整倍性与肿瘤发生密切相关。虽然人们多年来对非整倍性的发生进行了大量研究,但确切机制至今尚未阐明。非整倍性患儿和肿瘤发病往往给家庭和社会带来沉重负担,因此,深入探讨非整倍性形成的分子调控机制,对降低非整倍性患儿出生风险和肿瘤发生有重要意义。人类常染色体非整倍性约90%是卵母细胞成熟过程中减数分裂错误所致,卵母细胞成熟与胞质密切相关,而胞质中动力蛋白(Dynein)因独特的生理功能在染色体分离和纺锤体形成关卡中起重要作用。但是,目前,传统荧光成像方法对Dynein和微管蛋白以及染色体成像研究受到衍射极限的限制,分辨率较低,本研究尝试利用共聚焦显微和随机光学重建显微(random optical reconstruction microscopy,STORM)成像方法,观察卵母细胞减数分裂过程中动力蛋白、微管蛋白以及染色体的形态结构,为高空间分辨水平下研究卵母细胞非整倍性形成的分子调控机制提供了技术支持。针对此研究,本论文基于三维随机光学重建超分辨荧光显微镜开展研究工作,具体内容如下:1、介绍了本课题的研究背景,国内外现状及分析,重点介绍了远场超分辨荧光显微术,包括受激辐射损耗显微术、饱和结构光照明显微术、光激活定位显微术以及随机光重建显微术的工作原理以及优缺点。2、阐述了与本课题密切相关的随机光学重建超分辨荧光显微术的基本理论与技术问题,并详细介绍了实验室现有的自行搭建的三维超分辨荧光显微成像系统。3、研究了体细胞荧光超分辨成像的标记方法,挑选了微管结构丰富、伸展开来的小鼠成肌细胞(C2C12)、人胚胎成纤维细胞(MRC-5)和小鼠成纤维细胞(NIH-3T3)进行荧光标记,针对超分辨成像过程中出现的荧光分子不连续等问题,从固定液、抗体浓度以及抗体等方面进行优化,并获得了稳定的标记方法和成像条件。4、探索了卵母细胞荧光超分辨成像过程,首先构建了卵母细胞体外正常成熟体系和原钒酸钠(SOV)作用下的异常成熟体系,标记了减数分裂过程中两种重要蛋白以及染色体,通过共聚焦显微观察了Dynein分布、微管形成的纺锤体以及染色体形态与结构荧光定位,明确了减数分裂过程中不同时期共定位情况,随机光重建超分辨显微结果清晰的展现了纺锤体结构信息。5、最后进行了总结与展望,对论文内容进行了简单的总结和概括,并且根据现有实验结果,提出了优化实验设计的建议和展望。本研究的荧光显微成像结果表明,在正常成熟体系中,Dynein分布、微管形成的纺锤体以及染色体形态与结构荧光定位能反映卵母细胞发育的不同阶段,而在SOV作用下,纺锤体结构异常率增加,出现桶状、细长、团缩、杂乱、梨状、多极等典型异常纺锤体结构,Dynein与纺锤体定位基本一致,染色体结构异常率增加,出现掉队、散乱分布等异常情况。STORM显微结果更清晰的展现了纺锤体结构信息,以及异常纺锤体的紊乱情况,为高空间分辨水平下研究卵母细胞非整倍性形成机制提供了技术支持。
[Abstract]:Aneuploidy (aneuploidy) is the most common type of human chromosome abnormalities in reproductive cells, aneuploidy is the main reason causing human mental retardation, congenital malformations and pregnancy failure, somatic aneuploidy and tumorigenesis. Although for many years on the occurrence of aneuploidy were large study, but the exact mechanism has not been elucidated. Aneuploidy and cancer patients often brought a heavy burden on the family and society, therefore, in-depth study of the non molecular mechanism of aneuploidy formation, to reduce non significant aneuploidy and tumorigenesis. The risk of the birth of human chromosome aneuploidy about 90% is the error caused by meiosis during oocyte maturation, oocyte maturation and cytoplasm are closely related, and cytoplasmic dynein (Dynein) because of their unique physiological function in chromosome segregation and spindle formation level Play an important role. However, at present, the traditional fluorescence imaging method by the diffraction limit of Dynein and tubulin and chromosome imaging constraints, low resolution, this study attempts to use confocal microscopic and stochastic optical reconstruction (random optical reconstruction microscopy, STORM) imaging method, dynein observation of oocyte meiosis tubulin, and the morphology of chromosomes, molecular regulation mechanism of aneuploidy formation provides technical support for the high spatial resolution level of oocytes. In this research, the 3D stochastic optical reconstruction of super-resolution fluorescence microscopy to carry out research work based on the specific contents are as follows: 1, introduces the research background of this research and analysis of the situation at home and abroad, focuses on the far-field super-resolution fluorescence microscopy, including stimulated radiation loss of microscopic surgery, saturated structure The light was micro surgery, light activated localization microscopy and working principle of stochastic optical reconstruction microscopy as well as the advantages and disadvantages of.2, expounds the basic theory and technology of stochastic optical reconstruction is closely related with the subject of super-resolution fluorescence microscopy, and introduces the existing self built three-dimensional super-resolution fluorescence microscopy imaging system.3 study on somatic cell fluorescence labeling method of super resolution imaging, selection of microtubule structure rich stretch of mouse myoblasts (C2C12), human embryonic fibroblasts (MRC-5) and mouse fibroblast cells (NIH-3T3) labeled with fluorescence for super resolution fluorescence appeared in the imaging process of discontinuous problems from the fixed liquid, antibody concentration and antibody has been optimized, and the marking method and imaging condition of stable.4, explored oocyte fluorescence superresolution imaging process, the first First constructed in vitro oocyte maturation system and normal sodium orthovanadate (SOV) under the action of the abnormal mature system, marked the meiosis and chromosome two important proteins, by confocal microscopic observation of the Dynein distribution, the spindle microtubule formation and chromosome morphology and structure of positioning clear co localization of fluorescence. In the process of meiosis in different periods, random optical superresolution reconstruction results clearly show the structure of the spindle.5, finally summarizes and prospects, the contents of the paper are summarized and summarized, and according to the experimental results, and puts forward suggestions to optimize the experimental design. The results of fluorescence microscopy imaging show that the distribution of Dynein in the normal mature system, the formation of microtubules, spindle and chromosome morphology and structure of fluorescence localization can reflect the development of oocytes of different Stage, and in the presence of SOV spindle structure abnormal rate increased, barrel, slender, round shrink, messy, pear shaped, multi typical abnormal spindle structure, Dynein and spindle positioning consistent chromosomal structural abnormality rate increases, appear left behind, scattered and other anomalies.STORM microscopic results more clearly show the spindle the structure of information, and the disorder of abnormal spindle, high spatial resolution level of oocyte aneuploidy formation mechanism and provide technical support.

【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q253;TH742

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